JP6720194B2 - Method for producing L-serine using genetically modified microorganism lacking serine degradation pathway - Google Patents

Description

The present invention relates generally to the microbial industry and in particular to the production of L-serine using genetically modified bacteria. The present invention provides a transgenic microorganism, for example a bacterium in which the expression of a gene encoding an enzyme involved in the degradation of L-serine is attenuated by such as inactivation, which inactivation is particularly high. Suitable for producing L-serine in yield. The present invention also provides a method that allows microorganisms, and more particularly bacteria, to be resistant to higher concentrations of serine. The present invention also provides a method for producing L-serine or an L-serine derivative using such a genetically modified microorganism.

BACKGROUND OF THE INVENTION L-serine is an amino acid used in the cosmetic, pharmaceutical and medical industries. The estimated annual production of serine is 300-1000 tonnes (Leuchtenberger et al., 2005). This compound has been identified as one of the top 30 most interesting biochemicals because of its potential utility as a building block for biochemicals. Current production is based on the conversion of glycerin and methanol using resting cells (Hashishita et al., 1996), where the methylotroph converts methanol to formaldehyde and the CH ₂ -OH unit of the molecule is serine. It is converted to glycine using hydroxymethyltransferase (glyA). This fermentation process is time consuming and glycine is an expensive starting material. Therefore, development of a method for directly producing serine from glucose at low cost is attractive.

Serine has the potential to be fermented from glucose in very high theoretical yields (Burgard and Maranas, 2001). However, several challenges have to be addressed to increase yields, the most important being the degradation of serine in the producing organism. Serine has two key degradation pathways in E. coli. Carbonization of serine to pyruvate in E. coli is catalyzed by three deaminase, sdaA, sdaB and tdcG, whereas C. Glutamicum has only one deaminase (sdaA) that is active against serine. In both organisms, the conversion of serine to glycine is encoded by glyA. The production of serine by knockout of deaminase alone was performed in E. coli (Li et al., 2012) and C. Attempted in Glutamicum (Peters-Wendish et al., 2005). A transient accumulation of 1 mg/L glucose to 3.8 mg/L in E. coli was observed when only one of the pathway genes (serA) was overexpressed. C. Deletion of glutamicum deaminase is unimportant in serine titer and produces only a transient increase. In a recent study, E. coli was designed to increase the flux of 3-phosphoglycerate by perturbing the TCA cycle and the glycoxylate shunt (Gu et al., 2014). The resulting strain depleted in only one deaminase (sdaA) was reported to produce 8.45 g L-serine from 75 g/L glucose (11.2% yield).

C. Down-regulation of glyA in glutamicum (Peters-Wendlisch et al., 2005) produced 9 g/L serine from 40 g/L glucose, but produced an unstable strain. GlyA is an important enzyme that converts serine to glycine, and in this process one carbon unit is transferred to tetrahydrofolate (THF), which is used as a cofactor. Removal of the folate pathway as well as the supply of folic acid are stable C. It results in the production of glutamicum and 36 g/L serine, but with relatively low yields (Stolz et al., 2007).

The lack of both major serine degradation pathways (serine to pyruvate and serine to glycine) has not previously been achieved. Furthermore, serine is known to become virulent even at low concentrations in strains lacking the pyruvate degradation pathway (Zhang and Newman, 2008). Serine is expected to be able to inhibit the production of branched amino acids in E. coli (Hama et al., 1990), ie to inhibit the conversion of serine to hydroxypyruvate and acrylate, which are toxic to cells. (De Lorenzo, 2014). Therefore, for efficient production of L-serine or its derivatives, it is desirable to both remove the serine degradation pathway and address the problems associated with serine toxicity.

SUMMARY OF THE INVENTION It is an object of the present invention to provide means which allow a more efficient production of L-serine. More particularly, the object of the present invention is to provide a means enabling the production of L-serine with higher nominal yields and improved mass yields.

This task was achieved, for example, by the discovery that inactivation of genes encoding enzymes involved in the degradation of L-serine, in particular the genes sdaA, sdaB, tdcG and glyA, can increase L-serine production.

The invention thus provides in a first aspect a bacterium, in particular a bacterium capable of producing L-serine. The bacterium is then modified so as to attenuate the expression of the gene encoding the polypeptide having serine deaminase activity and the expression of the gene encoding the polypeptide having serine hydroxymethyl transferase activity.

More particularly, the present invention provides bacteria modified to attenuate the expression of the genes sdaA, sdaB, tdcG and glyA, for example by inactivating these genes.

In a second aspect, the present invention provides a method for producing L-serine, which comprises culturing the bacterium as described above in a culture medium.

The present invention provides in a further aspect yeasts, for example yeasts capable of producing Saccharomyces cerevisiae, in particular L-serine, wherein said yeast comprises a gene encoding a polypeptide having serine deaminase activity. It has been modified to attenuate expression and the expression of a gene encoding a polypeptide having serine hydroxymethyl transferase activity.

More specifically, the invention provides the yeast Saccharomyces cereviae, which has been modified to attenuate the expression of the genes CHA1, SHM1 and SHM2, for example by inactivating these genes.

In a further aspect, the present invention provides a method for producing L-serine or an L-serine derivative, which comprises culturing yeast as described above in a medium.

The invention in a further aspect is at least about 90%, eg at least about 93%, to an (isolated) nucleic acid molecule, for example the amino acid sequence set forth in SEQ ID NO: 11 having an amino acid substitution at position Y356, S357 and/or S359. %, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, a vector comprising a nucleotide sequence encoding a polypeptide having an amino acid sequence having sequence homology. provide.

The invention in a further aspect is at least about 90%, for example at least about 93%, at least about 95%, at least about at least about 90% amino acid sequence having an amino acid substitution at position Y356, S357 and/or S359. Provided are (isolated) polypeptides having amino acid sequences with 96%, at least about 97%, at least about 98%, or at least about 99% homology. The (isolated) polypeptide may be one (and isolated form) expressed by the bacterium of the present invention.

The present invention can be summarized by the following items:
1. A bacterium modified to attenuate expression of a gene encoding a polypeptide having serine deaminase activity and modified to attenuate expression of a gene encoding a polypeptide having serine hydroxymethyl transferase activity.

2. The bacterium according to item 1, wherein the expression of the genes sdaA, sdaB, tdcG and glyA is attenuated.

3. The bacterium according to item 1 or 2, wherein the expression of the gene is attenuated by the inactivation of the gene.

4. 4. The bacterium of any one of items 1 to 3, wherein the bacterium is further modified to overexpress 3-phosphoglycerate dehydrogenase, phosphoserine phosphatase and phosphoserine aminotransferase.

5. 5. The bacterium according to any one of items 1 to 4, wherein the bacterium comprises an exogenous nucleic acid molecule comprising a nucleotide sequence encoding 3-phosphoglycerate dehydrogenase.

6. 6. The bacterium of any one of paragraphs 1-5, wherein the bacterium comprises an exogenous nucleic acid molecule comprising a nucleotide sequence encoding a 3-foglycerin aminotransferase.

7. 7. The bacterium according to any one of items 1 to 6, wherein the bacterium comprises an exogenous nucleic acid molecule containing a nucleotide sequence encoding a phosphoserine phosphatase.

8. The bacterium according to any one of Items 5 to 7, wherein the exogenous nucleic acid molecule is an expression vector.

9. The bacterium according to any one of items 5 to 7, wherein the exogenous nucleic acid is stably introduced into the genome of the bacterium.

10. 10. The bacterium of any of paragraphs 1-9, wherein the bacterium is capable of growing in minimal medium containing L-serine at a concentration of at least about 6.25 g/L.

11. The bacterium can grow at a growth rate of at least 0.1 hr ⁻¹ during logarithmic growth in minimal medium containing L-serine at a concentration of at least about 6.25 g/L. The bacterium according to any one of items.

12. 12. The bacterium according to any one of items 1 to 11, wherein the bacterium is further modified to overexpress the gene ydeD.

13. 13. The bacterium according to any one of items 1 to 12, wherein the bacterium comprises an exogenous nucleic acid molecule comprising a nucleotide sequence encoding a protein product of the gene ydeD.

14. The bacterium according to item 13, wherein the exogenous nucleic acid is an expression vector.

15. The bacterium according to item 13, wherein the exogenous nucleic acid is stably introduced into the genome of the bacterium.

16. Said bacterium comprises in the thrA gene one or more nucleotide substitutions that result in an amino acid substitution at position Y356 in the encoded polypeptide, one or more nucleotide substitutions that result in an amino acid substitution at position S357 in the encoded polypeptide. And/or one or more nucleotide substitutions that result in an amino acid substitution at position S359 in the encoded polypeptide, wherein the substitution at position Y356 is Y356C, Y356T, Y356V, Y356S, Selected from the group consisting of Y356W, Y356Q, Y356G, Y356N, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356L and Y356L; Selected from the group consisting of S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; and the substitution at the S359 position is S359R, S359G, S359M, S359F, S359T, S359P, S359S, S359V, S359S, S359V, S359S. The bacterium of any one of items 1 to 15, selected from the group consisting of:, S359K, S359E and S359L.

17. The bacterium comprises in the thrA gene one or more nucleotide substitutions that result in an amino acid substitution at position Y356 in the encoded polypeptide, wherein the substitution at position Y356 is Y356C, Y356T, Y356V. , Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L, the bacterium according to any one of items 1 to 16. ..

18. The bacterium comprises in the thrA gene one or more nucleotide substitutions that result in an amino acid substitution at position S357 in the encoded polypeptide, wherein the substitution at position S357 is S357R, S357V, S357P. , S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M, The bacterium of any one of items 1 to 17, selected from the group consisting of:

19. The bacterium comprises one or more nucleotide substitutions within the thrA gene that result in amino acid substitutions at position S359 in the encoded polypeptide, wherein the substitution at position S359 is S359R, S359G, S359M. , S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L, the bacterium of any one of items 1-18.

20. 20. The bacterium comprises one or more nucleotide substitutions within the thrA gene that result in one or more amino acid substitutions selected from the group consisting of Y356C, S357R and S359R. The bacterium described in 1.

21. Any of Items 1-20, wherein the bacterium expresses an aspartate kinase I/homoserine dehydrogenase I (ThrA) mutant having one or more amino acid substitutions selected from the group consisting of Y356C, S357R and S359R. The bacterium according to Item 1.

22. The bacterium comprises a nucleotide sequence encoding a polypeptide having an amino acid sequence having at least about 90% sequence homology to the amino acid sequence set forth in SEQ ID NO:11 that includes amino acid substitutions at positions Y356, S357 and/or S359. The bacterium according to any one of items 1 to 15, comprising an exogenous nucleic acid molecule.

23. The substitution at the position of Y356 is selected from Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R, and Y356L, S356, and Y356L. The substitution at the position is selected from the group consisting of S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; the substitution at the S359 position is S359R, S359G, S359G. 23. The bacterium of item 22, selected from the group consisting of S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

24. The bacterium comprises an exogenous nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having an amino acid sequence having at least about 90% sequence homology to the amino acid sequence set forth in SEQ ID NO: 11 containing an amino acid substitution at position Y356. The bacterium according to any one of Items 1 to 23, which comprises:

25. The substitution at the position of Y356 includes Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R, Y356L, and Y356L. The bacterium according to 24.

26. The bacterium comprises an exogenous nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide having an amino acid sequence having at least about 90% sequence homology to the amino acid sequence set forth in SEQ ID NO: 11 that includes an amino acid substitution at position S357. The bacterium according to any one of Items 1 to 25.

27. 27. The bacterium of item 26, wherein the substitution at position S357 is selected from the group consisting of S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M.

28. The bacterium comprises an exogenous nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having an amino acid sequence having a sequence homology of at least about 90% with the amino acid sequence of SEQ ID NO: 11 containing an amino acid substitution at position S359. The bacterium according to any one of Items 1 to 25, which comprises:

29. 29. The bacterium of item 28, wherein the substitution at position S359 is selected from the group consisting of S359R, S359G, S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

30. The bacterium comprises one or more nucleotide substitutions in the Irp gene that result in an amino acid substitution (eg, amino acid substitution D143G), such as a non-conservative amino acid substitution at position D143 in the encoded polypeptide. 30. The bacterium according to any one of 1 to 29.

31. The bacterium comprises one or more nucleotide substitutions in the rho gene that result in an amino acid substitution, such as a non-conservative amino acid substitution at position R87 in the encoded polypeptide (eg, amino acid substitution R87L). The bacterium according to any one of 1 to 30.

32. The bacterium comprises one or more nucleotide substitutions in the eno gene that result in an amino acid substitution (eg, amino acid substitution V164L), such as a non-conservative amino acid substitution at position V164 in the encoded polypeptide. 32. The bacterium according to any one of 1 to 31.

33. The bacterium comprises one or more nucleotide substitutions in the argP gene that result in an amino acid substitution (eg, amino acid substitution Q132K), such as a non-conservative amino acid substitution at position Q132 in the encoded polypeptide. The bacterium according to any one of claims 1 to 32.

34. The bacterium comprises one or more nucleotide substitutions in the tufA gene that result in an amino acid substitution (eg, amino acid substitution G19V), such as a non-conservative amino acid substitution at position G19 in the encoded polypeptide. The bacterium according to any one of claims 1 to 33.

35. The bacterium comprises one or more nucleotide substitutions in the cycA gene that result in an amino acid substitution (eg, amino acid substitution I220V), such as a non-conservative amino acid substitution at position I220 in the encoded polypeptide. The bacterium according to any one of items 1 to 34.

36. The bacterium comprises one or more nucleotide substitutions in the rpe gene that result in an amino acid substitution (eg, amino acid substitution I202T), such as a non-conservative amino acid substitution at position I202 in the encoded polypeptide. The bacterium according to any one of items 1 to 35.

37. The bacterium comprises one or more nucleotide substitutions in the yojl gene that result in amino acid substitutions such as non-conservative amino acid substitutions at position D334 in the encoded polypeptide (eg, amino acid substitution D334H). 37. The bacterium according to any one of items 36 to 36.

38. The bacterium comprises one or more nucleotide substitutions in the hyaF gene that result in amino acid substitutions such as non-conservative amino acid substitutions at position V120 in the encoded polypeptide (eg, amino acid substitution V120G). 38. The bacterium according to any one of 37 to 37.

39. 39. The bacterium of any one of paragraphs 1 to 38, wherein the bacterium has been further modified to attenuate expression of the gene pykF (eg, by gene inactivation).

40. 40. The bacterium of any one of paragraphs 1 to 39, wherein the bacterium has been further modified (eg, by gene inactivation) to attenuate expression of the gene malT.

41. The bacterium comprises one or more nucleotide substitutions in the rpoB gene that result in amino acid substitutions such as non-conservative amino acid substitutions at position P520 in the encoded polypeptide (eg amino acid substitution P520L). The bacterium according to any one of claims 1 to 40.

42. The bacterium comprises one or more nucleotide substitutions in the fumB gene that result in an amino acid substitution (eg, amino acid substitution T218P), such as a non-conservative amino acid substitution at position T218 in the encoded polypeptide. 42. The bacterium according to any one of 41 to 41.

43. The bacterium comprises in the gshA gene one or more nucleotide substitutions that result from an amino acid substitution (eg, amino acid substitution A178V), such as a non-conservative amino acid substitution at position A178 in the encoded polypeptide. The bacterium according to any one of 1 to 42.

44. 44. The bacterium of any one of paragraphs 1-43, wherein the bacterium has been further modified to attenuate expression of the gene lamB (eg, by gene inactivation).

45. The bacterium has within its genome a reference genome E. coli deposited under NCBI accession number NC_000913.2. 45. A bacterium according to any one of items 1 to 44 comprising a deletion of about 2854 base pairs from a position corresponding to position 850092 in E. coli K12MG1655.

46. The deletion results in a deletion of the first 5 base pairs of rhtA, a complete deletion of genes ompX and ybiP, a deletion of 239 base pairs of sRNA rybA and a deletion of 77 base pairs of the mntS gene, item 45. Bacteria as described.

47. The bacterium has been identified in its genome as E. coli deposited under NCBI accession number NC_000913.2. 47. A bacterium according to any one of clauses 1 to 46, which comprises an insertion of an insert sequence element IS1 of 768 base pairs in length in the lagging strand at a position corresponding to position 3966174 in the E. coli K12MG1655 reference genome.

48. The bacterium has E. coli within its genome, which has been deposited with NCBI accession number NC_000913.2. 48. A bacterium according to any one of paragraphs 1 to 47, which comprises an insertion of a single base pair at a position corresponding to position 2942629 in the E. coli K12MG1655 reference genome.

49. The bacterium has E. coli within its genome, which has been deposited with NCBI accession number NC_000913.2. 49. A bacterium according to any one of items 1 to 48, which comprises the insertion of an insertion sequence element IS4 of 1342 base pairs in length at a position corresponding to position 2942878 in the E. coli K12MG1655 reference genome.

50. The bacterium has E. coli within its genome, which has been deposited with NCBI accession number NC_000913.2. 50. The bacterium of any one of paragraphs 1 to 49, which comprises an insertion of 1 base pair at a position corresponding to position 2599854 in the E. coli K12MG1655 reference genome.

51. The bacterium has E. coli within its genome, which has been deposited with NCBI accession number NC_000913.2. 52. A bacterium according to any one of clauses 1 to 51, which comprises an insertion of an insert sequence element IS1 of 768 base pairs in length in the lagging strand at a position corresponding to position 2492323 in the E. coli K12MG1655 reference genome.

52. The bacterium has E. coli within its genome, which has been deposited with NCBI accession number NC_000913.2. 52. A bacterium according to any one of paragraphs 1 to 51, which comprises the insertion of an insert sequence element IS5 of 1195 base pairs in length at a position corresponding to position 121518 in the E. coli K12MG1655 reference genome.

53. The bacterium has E. coli within its genome, which has been deposited with NCBI accession number NC_000913.2. 53. A bacterium according to any one of clauses 1 to 52, which comprises an insertion of an insert sequence element IS1 of 768 base pairs in length in the lagging strand at a position corresponding to position 1673670 in the E. coli K12MG1655 reference genome.

54. 54. The bacterium of any of paragraphs 1-53, wherein the bacterium has been modified to attenuate expression of a gene encoding a polypeptide having glucose 6-phosphate-1-dehydrogenase (G6PDH) activity.

55. 55. The bacterium of item 54, wherein the expression of the zwf gene is attenuated.

56. The bacterium according to item 54 or 55, wherein the expression of the gene is attenuated by inactivation of the gene.

57. 57. The bacterium according to any one of items 1 to 56, wherein the polypeptide expresses a polypeptide encoded by the brnQ gene, wherein said polypeptide ends at position 308 or any position upstream thereof. Bacteria.

58. The bacterium further expresses a polypeptide encoded by the thrA gene, wherein serine at position 357 in said polypeptide is replaced by another amino acid, eg arginine, any of items 54 to 57. The bacterium according to Item 1.

59. Any one of paragraphs 54 to 58, wherein said bacterium expresses a polypeptide encoded by the rho gene, wherein said polypeptide is substituted at position 87R by another amino acid, eg L. The bacterium according to the item.

60. The bacterium expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO:11, or at least about 90%, at least about 93%, at least about 95%, at least about 96 to the amino acid sequence set forth in SEQ ID NO:11. %, at least about 97%, at least about 98% or at least about 99% sequence homology is expressed, wherein said amino acid sequence is such that at position 357 serine is replaced by another amino acid, eg arginine. ing;
Expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 13 or at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97% amino acid sequence set forth in SEQ ID NO:13. %, at least about 98%, at least about 99% polypeptide having sequence homology is expressed, wherein said amino acid sequence is at position 87 with R replaced by another amino acid, eg L; Expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 32, or at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97% to the amino acid sequence set forth in SEQ ID NO: 32. The bacterium of any one of paragraphs 54 to 58, which expresses a polypeptide having a sequence homology of at least about 98%, at least about 99%.

61. 57. The bacterium of any one of paragraphs 1 to 56, wherein the bacterium has been further modified (eg, by gene inactivation) to attenuate expression of the gene brnQ.

62. The bacterium expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO:11, or at least about 90%, at least about 93%, at least about 95%, at least about 96 to the amino acid sequence set forth in SEQ ID NO:11. %, at least about 97%, at least about 98%, at least about 99%, a polypeptide having sequence homology is expressed, wherein said amino acid sequence is such that at position 357 serine is replaced by another amino acid, for example arginine. The bacterium according to item 61, which comprises:

63. The bacterium expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO:13, or at least about 90%, at least about 93%, at least about 95%, at least about 96% of the amino acid sequence set forth in SEQ ID NO:13. , A polypeptide having a sequence homology of at least about 97%, at least about 98%, at least about 99%, wherein said amino acid sequence is at position 87 with R substituted by another amino acid, for example L. The bacterium according to Item 61 or 62.

64. 64. The bacterium according to any one of Items 1 to 63, which belongs to the genus Escherichia coli.

65. The bacterium is Escherichia coli, Arsenophonus, Biostraticola, Brenneria, Buchnera, Budvicia aericella, Butvicea aericella, Buttiacera, Butchia terracella, Buttiauxella, Bacteria, Cutia, Butchia, Bacteria, Buttiauxella, Butchia, Bacteria, Buttiauxella, Butchia, Bacteria, Butchia, Bacteria, Buttiauxella, Butchia, Bacteria, Butchia, Bacteria ), Cronobacter, Dickeyea, Edwardsieella, Enterobacter, Erwinia, Ewingera Klebira, Gibbhafia, Gibbhafia, Gibbsifla, Gibbsierra (Leclercia), Reminorella (Leminorella), Lonsdalea (Lonsdalea), Mongrovibacter (Mangrovibacter), Moerella (Moellerella), Morganella (Morganella), Obesumbacteria (Obesumpaneta), Obesumbacteria (Obesumpactera, Obesumbacteria). , Phaseolibacter, Photorhabdus, Plesiomonas, Proteus, Rahnerella, Raohultellaon, Raoultella, Saccharoba, Saccharobacter (Saccharoba, Saccharobacter). , Serratia, Shimwellia, Sodalis, Tatumella, Torsellia, Trabulsiella, Wigglesworthia (Wilglesworthia and Wisglesia), Wigglesworthia (Wigglesworthia and Wierglesia). 65. The bacterium according to item 64, which belongs to the genus selected from the group consisting of:

66. 66. The bacterium according to item 64 or 65, which belongs to the genus Escherichia coli.

67. 67. The bacterium according to item 66, wherein the bacterium is Escherichia coli.

68. A method for producing L-serine or an L-serine derivative, the method comprising culturing the bacterium according to any one of items 1 to 67 in a medium. A method for producing a serine derivative.

69. The method according to Item 68, which is a method for producing L-serine.

70. The method according to item 68, which is a method for producing an L-serine derivative.

71. 69. The method of item 68, wherein the L-serine derivative is selected from the group consisting of L-cysteine, L-methionine, L-arginine, O-acetylserine, L-tryptophan, thiamine, ethanolamine and ethylene glycol.

72. 69. The method according to item 68, which is a method for producing L-cysteine.

73. The method according to item 68, which is a method for producing L-methionine.

74. The method according to item 68, which is a method for producing L-glycine.

75. 69. The method according to item 68, which is a method for producing O-acetylserine.

76. 69. A method according to item 68, which is a method for producing L-tryptophan.

77. 69. A method according to item 68, which is a method for producing thiamine.

78. 69. A method according to item 68, which is a method for producing ethanolamine.

79. 69. A method according to item 68, which is a method for producing ethylene glycol.

80. The method according to any one of Items 68 to 80, further comprising recovering L-serine or an L-serine derivative from the medium.

81. A yeast modified to attenuate expression of a gene encoding a polypeptide having serine deaminase activity and to attenuate expression of a gene encoding a polypeptide having serine hydroxymethyl transferase activity.

82. 82. The yeast according to item 81, wherein the expression of the genes CHA1, SHM1 and SHM2 is attenuated.

83. The yeast according to item 81 or 82, wherein expression of the gene is attenuated by inactivation of the gene.

84. 84. The yeast of any one of paragraphs 81-83, wherein the yeast is further modified to overexpress 3-phosphoglycerate dehydrogenase, phosphoserine phosphatase and phosphoserine aminotransferase.

85. The yeast according to any one of items 81 to 84, which belongs to the genus Saccharomyces.

86. The yeast according to item 85, which is Saccharomyces cereviae.

87. A method for producing L-serine or an L-serine derivative; comprising culturing the yeast according to any one of the following items 81 to 86 in a medium, Method of manufacturing.

88. (Isolated) a nucleic acid molecule, such as a vector, with at least about 90%, for example at least about 93%, to the amino acid sequence set forth in SEQ ID NO: 11 comprising amino acid substitutions at positions Y356, S357 and/or S359, A (isolated) nucleic acid comprising a nucleotide sequence encoding a polypeptide having an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence homology. molecule.

89. 89. The (isolated) nucleic acid molecule of item 88, wherein the polypeptide has the amino acid sequence set forth in SEQ ID NO: 11 that includes amino acid substitutions at positions Y356, S357 and/or S359.

90. 90. An (isolated) nucleic acid molecule according to item 88 or 89, wherein the amino acid substitution is at position Y356.

91. 91. The (isolated) nucleic acid molecule according to any one of items 88 to 90, wherein the amino acid substitution is at position S357.

92. 92. The (isolated) nucleic acid molecule according to any one of items 88 to 91, wherein the amino acid substitution is at position S359.

93. Substitutions at the Y356 position are selected from Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356S and 7356L. The substitution at position is selected from the group consisting of S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; and the substitution at S359 position is S359R, (Isolation) according to any one of items 88 to 92, selected from the group consisting of S359G, S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359C, S359K, S359E and S359L. Nucleic acid molecule.

94. At least about 90%, such as at least about 93%, at least about 95%, at least about 96%, at least about 97, to the amino acid sequence set forth in SEQ ID NO: 11 that includes amino acid substitutions at positions Y356, S357 and/or S359. %, at least about 98%, or at least about 99% sequence homology (isolated) polypeptide having an amino acid sequence.

95. 95. The (isolated) polypeptide according to item 94, wherein the polypeptide has the amino acid sequence set forth in SEQ ID NO: 11 comprising amino acid substitutions at positions Y356, S357 and/or S359.

96. The (isolated) polypeptide according to items 94 or 95, wherein the amino acid substitution is at position Y356.

97. Amino acid substitutions (isolated) polypeptide according to any one of items 94 to 96, which is at position S357.

98. Amino acid substitutions are (isolated) polypeptides according to any one of items 94 to 97, which are at position S359.

99. Substitutions at the Y356 position are selected from Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356S and 7356L. The substitution at the position is selected from the group consisting of S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; the substitution at the S359 position is S359R, S359G, S359G. The (isolated) polypeptide according to any one of items 94 to 98, selected from the group consisting of S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

FIG. 1 is a diagram showing deletion of major genes sdaA, sdaB, tdcG and glyA involved in serine degradation in Escherichia coli. FIG. 2 is a diagram showing a vector map of the construct. FIG. 3 is a diagram showing serine production during batch fermentation. FIG. 4 shows the growth profile using overexpression of the exporter ydeD in Q1(DE3). FIG. 5 shows the growth profile of Q1 and Q3 in 12.5 g L-serine. FIG. 6 is an adaptive laboratory evolution (ALE) experiment to improve resistance to serine. (A) Growth rate during evolution testing. (B) shows improved growth of related strains in the presence of high concentrations of serine. FIG. 7 shows the efficiency of mutations in thrA in resistance to serine. FIG. 8 shows the identification of mutations that cause increased resistance to serine. FIG. 9 is a diagram showing serine production and cell density (OD600 nm) measured at various time points during fed-batch fermentation of (A) Q1(DE3) and Q3(DE3) strains. FIG. 10: Cell density and serine titer of (A) ALE8-8(DE3) strain. It is a figure which shows the mass yield from (B) glucose. FIG. 11 shows mutant E. coli with the various amino acid substitutions observed at each position 356(11A), 357(11B) and 359(11C) of ThrA (each amino acid substitution is shown in single letter code). It is a figure which shows the growth rate of a strain.

The present invention will be specifically described below.

DETAILED DESCRIPTION OF THE INVENTION Unless otherwise specified herein, all industrial and scientific terms used are commonly understood by one of ordinary skill in the art of biochemistry, genetics, and microbiology. Have the same meaning.

All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention using the appropriate methods and materials described herein. All publications, patent specifications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will prevail. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting unless otherwise stated.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology and recombinant DNA, which are well within the skill of those in the art. It is within the range of skills. Such techniques are explained fully in the literature. For example, current protocols in molecular biology (Frederick M.AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: Laboratory Manual, Third Edition (Sambrook et al., 2001, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Publications); Oligonucleotide Synthesis (MJ Gait Editor, 1984); Mullis et al., US Pat. No. 4,683,195; Nucleic Acid Hybridization (BD Harris & SJ Higgins et al., 1984). Transcription and translation (BD Hames & S. J. Higgins editor, 1984); Culture of animal cells (RI Freshney, Alan R. Liss, Inc., 1987); Immobilized cells and enzymes (IRL). Press, 1986); Perbal, Guidelines for Molecular Cloning (1984); Methods in Immunology (series) (J. Abelson and M. Simon, Editor-in-Chief, Academic Publishing Co., NY), especially 154 and 155 (Wu et al., Editor). And 185, "Gene Expression Technology" (D. Goeddel Editor); Gene Transfer Vectors for Mammalian Cells (JH Miller and MP Calos Editor, 1987, Cold Spring Harbor Laboratory); Cells and Molecules. Immunochemistry in Biology (Mayer and Walker, Editor, Academic Publications, London, 1987); Experimental Immunology References, Volumes 1 to 4 (Editor DM Weir & CC Blackwell, 1986); and See Treatment of Mouse Embryos (Cold Spring Harbor Laboratory Publications, Cold Spring Harbor, NY, 1986).

Bacteria of the Present Invention As described above, the present invention provides that the production of L-serine is achieved by, for example, inactivating the genes encoding the enzymes involved in the degradation of L-serine, especially the genes sdaA, sdaB, tdcG and glyA. It is specifically based on the finding that it can be increased.

Accordingly, the present invention provides a bacterium, in particular a bacterium capable of producing L-serine, wherein said bacterium attenuates the expression of a gene encoding a polypeptide having serine deaminase activity and It has been modified to attenuate expression of a gene encoding a polypeptide having hydroxymethyltransferase activity.

More particularly, the invention provides bacteria modified to attenuate expression of the genes sdaA, sdaB, tdcG and glyA.

The genes sdaA, sdaB, tdcG encode L-serine deaminase I (SdaA), L-serine deaminase II (SdaB) and L-serine deaminase III (TdcG), respectively, which are one step in the L-serine degradation pathway. It is the only three enzymes that do this and convert serine to the basic cellular building block, pyruvate. For example, further information regarding E. coli sdaA, sdaB, tdcG is available in EcoCyc (www.biocyc.org) under accession numbers EG10930, EG11623 and G7624, respectively. Representative nucleotide sequences for sdaA, sdaB and tdcG are set forth in SEQ ID NOs: 1-3, respectively.

The gene glyA encodes serine hydroxymethyl transferase (GlyA), which converts serine to glycine, converts the methyl group to tetrahydrofolate, thus forming 5,10-methylene-tetrahydrofolate (5,10-mTHF). .. 5,10-mTHF is the major source of C1 units in cells, making GlyA a key enzyme in the biosynthesis of purines, thymidine, methionine, choline and fat. Further information on glyA is available on EcoCyc (www.biocyc.org) under accession number EG10408. A representative nucleotide sequence for glyA is set forth in SEQ ID NO:4.

Gene expression may be attenuated by gene inactivation. Therefore, the bacterium according to the present invention has been modified to inactivate a gene encoding a polypeptide having serine deaminase activity and inactivate expression of a gene encoding a polypeptide having serine hydroxymethyl transferase activity. It may be one. According to a particular embodiment, the expression of the genes sdaA, sdaB, tdcG and glyA is attenuated by inactivation of these genes. Therefore, the bacterium according to the present invention can be modified so as to inactivate the genes sdaA, sdaB, tdcG and glyA.

By introducing a mutation into a gene on the chromosome, the expression of the gene can be reduced so that the intracellular activity of the protein encoded by the gene is reduced as compared to the non-denatured strain. Mutations that result in a diminished expression of a gene include one nucleotide or more exchange that results in one amino acid substitution in the protein encoded by the gene (missense mutation), introduction of a stop codon (nonsense mutation). , Deletions or insertions of nucleotides that cause frameshifts, insertions of drug resistance genes, or deletions of some or all of the genes (Qiu and Goodman, 1997; Kwon et al., 2000). Expression can also be attenuated by denaturation of expression control sequences such as promoters, Shine-Dalgarno (SD) sequences (WO95/34672).

For example, the following method may be used to introduce a mutation by genetic recombination. Mutant genes encoding mutant proteins with reduced activity can also be prepared. Therefore, the native gene on the chromosome is replaced by the mutant gene by homologous recombination and the resulting strain can be selected. Gene replacement using homologous recombination is by using linear DNA, which is known as "lamda-red mediated gene replacement" (Datsenko and Wanner, 2000), or temperature. It can be carried out by using a plasmid containing a sensitive origin of replication (US Pat. No. 6,303,383, or JP05-007491A). In addition, site-directed mutations due to gene replacement can also be inserted using homologous recombination as described above with plasmids that are not replicable in the host.

By inserting a transposon or IS factor into the coding region of the gene (US Pat. No. 5,175,107) or by conventional methods such as UV irradiation or nitrosoguanidine (N-methyl-N′-nitro-N-nitrosoguanidine). Depending on the mutation used, site-directed mutagenesis, gene disruption using homologous recombination and/or gene exchange (Yu et al., 2000; and Datsenko and Wanner, 2000), eg, "lambda-red mediated gene exchange" Expression can also be attenuated. Lambda red mediated gene exchange is particularly suitable for the method of inactivating one or more genes described herein. Thus, in a specific embodiment, gene expression is attenuated by gene inactivation using lambda red-mediated gene exchange.

As shown in Figure 3, inactivation of all four genes involved in serine degradation (sdaA, sdaB, tdcG and glyA) is involved in L-serine degradation via the serine to pyruvate pathway. Glucose yields the highest specific productivity and highest yield from glucose compared to the inactivity of only three genes (sdaA, sdaB and tdcG).

Serine uses three enzymes encoded by the genes serA (encodes 3-phosphoglycerate dehydrogenase), serB (encodes phosphoserine phosphatase), and serC (encodes phosphoserine aminotransferase) for glycerine. Made from aldehyde-3-phosphate. These genes may be overexpressed to increase L-serine production. For example, information relating to E. coli serA, serB and serC is available in EcoCyc (www.biocyc.org) under registration numbers EG10944, EG10945 and EG10946, respectively.

Therefore, according to a particular embodiment, the bacterium has been modified to overexpress 3-phosphoglycerate dehydrogenase, phosphoserine phosphatase and phosphoserine aminotransferase. More specifically, the bacterium has been further modified to overexpress the genes serA, serB and serC. This includes one or more (eg 2 or 3) exogenous nucleic acid molecules in bacteria, for example a nucleotide sequence encoding 3-phosphoglycerate dehydrogenase, a nucleotide sequence encoding phosphoserine phosphatase and/or Alternatively, it may be achieved by inserting one or more vectors containing a nucleotide sequence encoding a phosphoserine aminotransferase.

The 3-phosphoglycerate dehydrogenase may be derived from the same species as the bacterium in which it is overexpressed, or from a different species than it is overexpressed (ie, it is heterologous is there). In a particular embodiment, the 3-phosphoglycerate dehydrogenase is derived from the same species in which it is overexpressed. In certain other embodiments, the 3-phosphoglycerate dehydrogenase is derived from a different species than it is overexpressed (ie, it is heterologous).

In a particular embodiment, the bacterium comprises an exogenous nucleic acid molecule comprising a nucleotide sequence encoding a 3-phosphoglycerate dehydrogenase. An exogenous nucleic acid molecule may include a nucleotide sequence that encodes a popeptide that includes the amino acid sequence set forth in SEQ ID NO:5. The exogenous nucleic acid molecule comprises at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the amino acid sequence set forth in SEQ ID NO:5. It may contain a nucleotide sequence encoding a polypeptide containing an amino acid sequence having sequence homology. Advantageously, the polypeptide has 3-phosphoglycerate dehydrogenase activity. More advantageously, the polypeptide has a 3-phosphoglycerate dehydrogenase activity similar to that of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:5.

The exogenous nucleic acid molecule has at least about 95%, for example at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 96%, at least about 95% of the amino acid sequence set forth in SEQ ID NO:5. It may include a nucleotide sequence that encodes a polypeptide that includes an amino acid sequence that has 97%, at least about 98%, or at least about 99% sequence homology. Advantageously, the polypeptide has 3-phosphoglycerate dehydrogenase activity. More advantageously, the polypeptide has a 3-phosphoglycerate dehydrogenase activity similar to that of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:5. The exogenous nucleic acid molecule may comprise a nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:5, wherein one or more, eg, about 1 to about 50, About 1 to about 40, about 1 to about 35, about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, 1 to about 5, or about 1 to about 3 amino acid residues have been substituted, deleted and/or inserted. Advantageously, the polypeptide has 3-phosphoglycerate dehydrogenase activity. More advantageously, the polypeptide has a 3-phosphoglycerate dehydrogenase activity similar to that of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:5. The exogenous nucleic acid molecule may include nucleotides encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:5, wherein about 1 to about 5 such as about 1 to about 3 Amino acid residues of have been substituted, deleted and/or inserted. Advantageously, the polypeptide has 3-phosphoglycerate dehydrogenase activity. More advantageously, the polypeptide has a 3-phosphoglycerate dehydrogenase activity similar to that of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:5.

A further advantage of overexpressing the mutant serA gene encoding 3-phosphoglycerate dehydrogenase is its resistance to feedback inhibition of serine. This may be achieved, for example, by deleting the last four C-terminal residues of wild-type 3-phosphoglycerate dehydrogenase (SerA). Alternatively, the feedback inhibition of SerA can be eliminated by a mutation that makes three residues H344, N346 and N364 alanine. A representative amino acid sequence of such a 3-phosphoglycerate dehydrogenase mutant is set forth in SEQ ID NO:6. Thus, according to a specific embodiment, the bacterium comprises an exogenous nucleic acid molecule comprising a nucleotide sequence encoding a 3-phosphoglycerate dehydrogenase that is resistant to feedback inhibition of serine. The exogenous nucleic acid molecule may include a nucleotide sequence that encodes a polypeptide that includes the amino acid sequence set forth in SEQ ID NO:6. The exogenous nucleic acid molecule comprises at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the amino acid sequence set forth in SEQ ID NO:6. It may contain a nucleotide sequence encoding a polypeptide containing an amino acid sequence having sequence homology. Advantageously, the polypeptide has 3-phosphoglycerate dehydrogenase activity. More advantageously, the polypeptide has a 3-phosphoglycerate dehydrogenase activity similar to that of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:6. The exogenous nucleic acid molecule comprises at least about 90%, at least about 93%, such as at least about 95%, such as at least about 96%, at least about 97%, at least about 98%, or at least the amino acid sequence set forth in SEQ ID NO:6. It may include nucleotide sequences with about 99% sequence homology. Advantageously, the polypeptide has 3-phosphoglycerate dehydrogenase activity. More advantageously, the polypeptide has a 3-phosphoglycerate dehydrogenase activity similar to that of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:6. The exogenous nucleic acid molecule may comprise a nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:6, wherein one or more, eg, about 1 to about 50, About 1 to about 40, about 1 to about 35, about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 1 to about 5, or about 1 to about 3 amino acid residues have been substituted, deleted and/or inserted. Advantageously, the polypeptide has 3-phosphoglycerate dehydrogenase activity. More advantageously, the polypeptide has a 3-phosphoglycerate dehydrogenase activity similar to that of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:6. The exogenous nucleic acid molecule may include a nucleotide sequence that encodes a polypeptide that includes the amino acid sequence set forth in SEQ ID NO:6, wherein about 1 to about 5, such as about 1 to about 3, Amino acid residues have been substituted, deleted and/or inserted. Advantageously, the polypeptide has 3-phosphoglycerate dehydrogenase activity. More advantageously, the polypeptide has a 3-phosphoglycerate dehydrogenase activity similar to that of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:6.

The phosphoserine phosphatase may be derived from the same species as the bacterium in which it is overexpressed, or from a different species than it is overexpressed (ie, it is heterologous). In a particular embodiment, the phosphoserine phosphatase is derived from the same species in which it is overexpressed. In certain other embodiments, the phosphoserine phosphatase is derived from a different species than it is overexpressed (ie, it is heterologous).

According to a particular embodiment, the bacterium comprises an exogenous nucleic acid molecule comprising a nucleotide sequence encoding a phosphoserine phosphatase. The exogenous nucleic acid molecule may include a nucleotide sequence that encodes a polypeptide that includes the amino acid sequence set forth in SEQ ID NO:7. The exogenous nucleic acid molecule comprises at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the amino acid sequence set forth in SEQ ID NO:7. It may contain a nucleotide sequence encoding a polypeptide containing an amino acid sequence having sequence homology. Advantageously, the polypeptide has phosphoserine phosphatase activity. More advantageously, the polypeptide has a 3-phosphoserine phosphatase activity similar to that of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:7. The exogenous nucleic acid molecule may comprise a nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:7, wherein one or more, eg, about 1 to about 50, About 1 to about 40, about 1 to about 35, about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, 1 to about 5, or about 1 to about 3 amino acid residues have been substituted, deleted and/or inserted. Advantageously, the polypeptide has phosphoserine phosphate activity. More advantageously, the polypeptide has a phosphoserine phosphatase activity similar to that of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:7. The exogenous nucleic acid molecule may include a nucleotide sequence that encodes a polypeptide that includes the amino acid sequence set forth in SEQ ID NO: 7, wherein about 1 to about 5, such as about 1 to about 3, Amino acid residues have been substituted, deleted and/or inserted. Advantageously, the polypeptide has phosphoserine phosphatase activity. More advantageously, the polypeptide has a phosphoserine phosphatase activity similar to that of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:7.

The phosphoserine aminotransferase may be derived from the same species as the bacterium in which it is overexpressed or from a different species than it is overexpressed (ie it is heterologous) .. In a particular embodiment, the phosphoserine aminotransferase is derived from the same species as the bacterium in which it is overexpressed. According to certain other embodiments, the phosphoserine aminotransferase may be derived from a different species than it is overexpressed (ie, it is heterologous).

In a particular embodiment, the bacterium comprises an exogenous nucleic acid molecule comprising a nucleotide sequence encoding a phosphoserine aminotransferase. The exogenous nucleic acid molecule may include a nucleotide sequence that encodes a polypeptide that includes the amino acid sequence set forth in SEQ ID NO:8. The exogenous nucleic acid molecule comprises at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the amino acid sequence set forth in SEQ ID NO:8. It may contain a nucleotide sequence encoding a polypeptide containing an amino acid sequence having sequence homology. Advantageously, the polypeptide has phosphoserine aminotransferase activity. More advantageously, the polypeptide has a phosphoserine aminotransferase activity similar to that of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:8. The exogenous nucleic acid molecule may comprise a nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:8, wherein one or more, eg, about 1 to about 50, About 1 to about 40, about 1 to about 35, about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, 1 to about 5, or about 1 to about 3 amino acid residues have been substituted, deleted and/or inserted. Advantageously, the polypeptide has phosphoserine aminotransferase activity. More advantageously, the polypeptide has a phosphoserine aminotransferase activity similar to that of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:8. The exogenous nucleic acid molecule may include a nucleotide sequence that encodes a polypeptide that includes the amino acid sequence set forth in SEQ ID NO:8, wherein about 1 to about 5, such as about 1 to about 3, Amino acid residues have been substituted, deleted and/or inserted. Advantageously, the polypeptide has phosphoserine aminotransferase activity. More advantageously, the polypeptide has phosphoserine aminotransferase properties similar to that of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:8.

Bacteria, eg, to attenuate expression of a gene encoding a polypeptide having serine deaminase activity, and to attenuate expression of a gene encoding a polypeptide having hydroxymethyltransferase (eg, gene inactivation). Denatured E. coli shows low resistance to serine. Therefore, it is desirable to provide bacteria that exhibit increased resistance to serine.

In this regard, the inventor has found that by evolving the bacterium by random mutation and by adaptive evolution, the overexpression of the novel exporter can reduce the toxicity of the product. As a result, bacteria are provided that have increased resistance to serine. As used herein, "increased resistance" refers to the ability of bacteria to grow in minimal medium containing L-serine at a concentration of at least about 6.25 g/L (eg, M9 minimal medium). Means

In certain embodiments, the bacterium of the invention comprises a minimal medium (eg, M9 minimal medium) comprising L-serine at a concentration of at least about 6.25 g/L (eg, at least about 12.5 g/L). ) Can grow. In a specific embodiment, the bacterium grows in minimal medium (eg, M9 minimal medium) containing L-serine at a concentration of at least about 12.5 g/L (eg, at least about 25 g/L). it can. In a specific embodiment, the bacterium can be grown in minimal medium (eg, M9 minimal medium) containing L-serine at a concentration of at least about 25 g/L (eg, at least about 40 g/L). According to a particular embodiment, the bacterium can be grown in minimal medium (eg M9 minimal medium) containing L-serine at a concentration of at least about 40 g/L (eg at least about 50 g/L). In a specific embodiment, the bacterium can be grown in a minimal medium (eg, M9 minimal medium) containing L-serine at a concentration of at least about 75 g/L (eg, at least about 100 g/L). According to a particular embodiment, the bacteria can be grown in minimal medium containing L-serine at a concentration of at least about 100 g/L (eg M9 minimal medium).

Advantageously, a minimal medium such as M9 minimal medium is supplemented with 2 mM glycine and 2 g/L glucose. Bacteria are generally cultivated at about 37° C. for about 24 to about 40 hours using full aeration.

In certain embodiments, the bacterium of the invention has a minimal medium (eg, M9 minimal medium) comprising L-serine at a concentration of at least about 6.25 g/L (eg, at least about 12.5 g/L). ), at a growth rate of at least about 0.1 hr ⁻¹ during logarithmic growth. In a specific embodiment, the bacterium of the invention is a minimal medium (eg, M9 minimal medium) comprising L-serine at a concentration of at least about 12.5 g/L (eg, at least about 25 g/L). In particular, it can grow at a growth rate of at least about 0.1 hr ⁻¹ during logarithmic growth. In a specific embodiment, the bacterium of the invention is in a minimal medium (eg, M9 minimal medium) containing L-serine at a concentration of at least about 25 g/L (eg, at least about 50 g/L). , Can grow at a growth rate of at least about 0.1 hr ⁻¹ during logarithmic growth.

Advantageously, minimal medium, eg M9 minimal medium, is supplemented with 2 mM glycine and 2 g/L glucose. Bacteria are generally cultivated at 37°C for about 24 to about 40 hours at 37°C using sufficient aeration.

In a specific embodiment, the bacterium of the invention comprises a minimal medium (eg, M9 minimal medium) containing L-serine at a concentration of at least about 25 g/L (eg, at least about 50 g/L), It can grow at a growth rate of at least about 0.1 hr ⁻¹ during logarithmic growth.

A novel exporter with improved resistance to serine when overexpressed in bacteria is the O-acetylserine/cysteine export protein encoded by the gene ydeD. Further information regarding E. coli ydeD is available, for example, at EcoCyc (www.biocyc.org) under accession number EG11639. A representative amino acid sequence of such an exporter protein is set forth in SEQ ID NO:9. Accordingly, the present invention provides a bacterium modified to overexpress the gene ydeD.

In a particular embodiment, the bacterium of the invention comprises an exogenous nucleic acid molecule such as an expression vector comprising a nucleotide sequence encoding the protein product of the gene ydeD. According to a particular embodiment, the bacterium comprises an exogenous nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:9.

The exogenous nucleic acid molecule comprises at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the amino acid sequence set forth in SEQ ID NO:9. It may contain a nucleotide sequence encoding a polypeptide containing an amino acid sequence having sequence homology. Advantageously, the polypeptide has O-acetylserine and/or cysteine transporter activity. More advantageously, the polypeptide has O-acetylserine and/or cysteine transporter activity similar to that of the polypeptide comprising the amino acid sequence set forth in SEQ ID NO:9. The exogenous nucleic acid molecule comprises an amino acid sequence that has about 95% sequence homology to the amino acid sequence set forth in SEQ ID NO: 9, such as at least about 96%, at least about 97%, at least about 98%, or at least about 99%. May include a nucleotide sequence encoding the polypeptide. Advantageously, the polypeptide has O-acetylserine and/or cysteine transporter activity. More advantageously, the polypeptide has O-acetylserine and/or cysteine transporter activity similar to that of the polypeptide comprising the amino acid sequence set forth in SEQ ID NO:9. The exogenous nucleic acid molecule may comprise a nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:9, wherein one or more, eg, about 1 to about 50, About 1 to about 40, about 1 to about 35, about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, 1 to about 5, or about 1 to about 3 amino acid residues have been substituted, deleted and/or inserted. Advantageously, the polypeptide has O-acetylserine and/or cysteine transporter activity. More advantageously, the polypeptide has O-acetylserine and/or cysteine transporter activity similar to that of the polypeptide comprising the amino acid sequence set forth in SEQ ID NO:9. The exogenous nucleic acid molecule may include a nucleotide sequence that encodes a polypeptide that includes the amino acid sequence set forth in SEQ ID NO: 9, wherein about 1 to about 5, such as about 1 to about 3, Amino acid residues have been substituted, deleted and/or inserted. Advantageously, the polypeptide has O-acetylserine and/or cysteine transporter activity. More advantageously, the polypeptide has O-acetylserine and/or cysteine transporter activity similar to that of the polypeptide comprising the amino acid sequence set forth in SEQ ID NO:9.

A modified YdeD polypeptide containing an additional extension of 6 histidine residues at the C-terminus is set forth in SEQ ID NO:10. In a specific embodiment, the bacterium comprises an exogenous nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:10. The exogenous nucleic acid molecule comprises at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the amino acid sequence set forth in SEQ ID NO:10. It may contain a nucleotide sequence encoding a polypeptide containing an amino acid sequence having sequence homology. Advantageously, the polypeptide has O-acetylserine and/or cysteine transporter activity. More advantageously, the polypeptide has O-acetylserine and/or cysteine transporter activity similar to that of the polypeptide comprising the amino acid sequence set forth in SEQ ID NO:10. The exogenous nucleic acid molecule comprises an amino acid sequence having at least about 95% sequence homology to the amino acid sequence set forth in SEQ ID NO: 10, such as at least about 96%, at least about 97%, at least about 98% or at least about 99%. May include a nucleotide sequence encoding the polypeptide. Advantageously, the polypeptide has O-acetylserine and/or cysteine transporter activity. More advantageously, the polypeptide has O-acetylserine and/or cysteine transporter activity similar to that of the polypeptide comprising the amino acid sequence set forth in SEQ ID NO:10. The exogenous nucleic acid molecule may include a nucleotide sequence that encodes a polypeptide that includes the amino acid sequence set forth in SEQ ID NO: 10, wherein one or more, eg, about 1 to about 50, About 1 to about 40, about 1 to about 35, about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, 1 to about 5, or about 1 to about 3 amino acid residues have been substituted, deleted and/or inserted. Advantageously, the polypeptide has O-acetylserine and/or cysteine transporter activity. More advantageously, the polypeptide has O-acetylserine and/or cysteine transporter activity similar to that of the polypeptide comprising the amino acid sequence set forth in SEQ ID NO:10. The exogenous nucleic acid molecule may include a nucleotide sequence that encodes a polypeptide that includes the amino acid sequence set forth in SEQ ID NO: 10, wherein about 1 to about 5, such as about 1 to about 3, Amino acid residues have been substituted, deleted and/or inserted. Advantageously, the polypeptide has O-acetylserine and/or cysteine transporter activity. More advantageously, the polypeptide has O-acetylserine and/or cysteine transporter activity similar to that of the polypeptide comprising the amino acid sequence set forth in SEQ ID NO:10.

As shown in Figure 4, growth of bacteria such as E. coli lacking the major serine degradation pathway significantly inhibits growth even in the presence of low concentrations of serine. Upon overexpression of ydeD, resistance to serine was also substantially increased, suggesting that YdeD may be transporting serine from the cell.

Bacteria of the present invention having improved resistance to serine, such as those capable of growing in minimal medium containing L-serine at a concentration of at least about 6.25 g/L as described above, are selected by random mutation or It can be obtained by adaptive evolution. Details of each are provided in Examples 4 and 5, respectively.

For example, adaptive evolution may be achieved by performing the following method: Prior to the start of the experiment, fill the appropriate tubes with 25 ml of medium, which is kept at 37° C. in a heat block. Controlled aeration was obtained using a magnetic tumble stirrer installed in the tube and rotating at 1800 rpm. At the start of the experiment, a single colony of (starting strain) was grown overnight in one of the tubes and 100 μL aliquots were used to seed tubes containing 25 ml of fresh medium. As the bacteria grew, multiple OD measurements were taken at 600 nm. Growth rates were calculated by taking the slope of the least-squares linear regression line that fits the log of the OD measurements. Once the target OD reached 0.4, 100 μl of medium was used to seed a new tube containing 25 ml of medium. Thus, after reaching the target cell density such that stationary phase was never reached, the culture was continuously (2 to 3 times a day) passed through tubes containing fresh medium. The experiment was started with an L-serine concentration of 3 g L-serine and subsequently increased to 6 g/L L-serine after reaching the desired growth rate. Once the population achieved a stable phenotype (ie growth rate), the concentration of L-serine was increased to 12 g/L. This process was repeated using L-serine 24, 50, 75 and 100 g/L. The final population may then be plated on LB-Agar and further L-serine resistant strains selected for further culture. Prior methods may be performed using an automated system that allows for breeding of populations that evolve manually or over several days while monitoring their growth rate.

As further demonstration herein, the inventor has identified beneficial mutations in many genes that are convinced of resistance to L-serine. The genes and mutations for each are listed in Table S5.

One such gene is thrA, which encodes aspartate kinase I/homoserine dehydrogenase I (ThrA). Further information on, for example, E. coli thrA is available at EcoCyc (www.biocyc.org) under accession number EG10998. A representative amino acid sequence of wild-type aspartate kinase I/homoserine dehydrogenase I (ThrA) is set forth in Sequence 11. As a demonstration of Examples 6 and 10, inserting a specific mutation in the amino acid sequence of aspartate kinase I/homoserine dehydrogenase resulted in a very marked increase in resistance to serine (Figures 7 and 11). In particular, the following mutations have been shown to be advantageous: Y356C, S357R and S359R.

According to a particular embodiment, the invention provides a bacterium expressing an aspartate kinase I/homoserine dehydrogenase I (ThrA) mutant that is not inhibited by L-serine.

According to a particular embodiment, the present invention provides a bacterium comprising in the thrA gene one or more nucleotide substitutions that result in one or more amino acid substitutions with increased resistance to L-serine. More specifically, it contains one or more nucleotide substitutions in the thrA gene that result in one or more amino acid substitutions in the polypeptide encoded at a position selected from the group consisting of Y356, S357 and S359. Bacteria are provided. Thus, the bacterium of the present invention may express aspartate kinase I/homoserine dehydrogenase I (ThrA) having one or more amino acid substitutions at a position selected from the group consisting of Y356, S357 and S359. More specifically, the bacterium of the present invention expresses a polypeptide having an amino acid sequence set forth in SEQ ID NO: 11 containing one or more amino acid substitutions at a position selected from the group consisting of Y356, S357 and S359. You may. Advantageously, the one or more amino acid substitutions are non-conservative substitutions.

According to a particular embodiment, the invention provides a bacterium comprising in the thrA gene one or more nucleotide substitutions which result in an amino acid substitution at position Y356 in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises an amino acid substitution at position Y356. According to a specific embodiment, the amino acid substitution consists of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L. To be selected. According to a specific embodiment, the amino acid substitution is selected from the group consisting of Y356C, Y356T, Y356V, Y356W, Y356Q, Y356G, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L. In a more specific embodiment, the amino acid substitutions are selected from the group consisting of Y356C, Y356T, Y356W, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L.

In another more specific embodiment, the amino acid substitutions are selected from the group consisting of Y356C, Y356W, Y356F, Y356I, Y356P, Y356R and Y356L.

In a particular embodiment, the bacterium comprises within the thrA gene one or more nucleotide substitutions that result in a Y356C substitution in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises a Y356C substitution. In a particular embodiment, the bacterium comprises in the thrA gene one or more nucleotide substitutions that result in a Y356T substitution in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises a Y356T substitution.

In a particular embodiment, the bacterium comprises one or more nucleotide substitutions that result in a Y356V substitution in the polypeptide encoded in the thrA gene. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises a Y356V substitution. According to a particular embodiment, one or more nucleotide substitutions that result in a Y356S substitution in the polypeptide encoded in the thrA gene are included. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises a Y356S substitution.

According to a particular embodiment, the bacterium comprises one or more nucleotide substitutions in the polypeptide encoded in the thrA gene that result in a Y356W substitution. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises a Y356W substitution. According to a particular embodiment, the bacterium comprises one or more nucleotide substitutions in the polypeptide encoded in the thrA gene that result in a Y356G substitution. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises a Y356G substitution. In a particular embodiment, the bacterium comprises in the thrA gene one or more nucleotide substitutions that result in a Y356N substitution in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the Y356N substitution. In a particular embodiment, the bacterium comprises in the thrA gene one or more nucleotide substitutions that result in a Y356D substitution in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises a Y356D substitution. In a particular embodiment, the bacterium comprises in the thrA gene one or more nucleotide substitutions that result in a Y356E substitution in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the Y356E substitution.

In a particular embodiment, the bacterium comprises in the thrA gene one or more nucleotide substitutions that result in a Y356F substitution in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises a Y356F substitution.

In a particular embodiment, the bacterium comprises one or more nucleotide substitutions that result in a Y356A substitution in the polypeptide encoded in the thrA gene. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises a Y356I substitution. In a particular embodiment, the bacterium comprises one or more nucleotide substitutions that result in a Y356A substitution in the polypeptide encoded in the thrA gene. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the Y356I substitution.

According to a particular embodiment, one or more nucleotide substitutions that result in a Y356P substitution in the polypeptide encoded in the bacterial thrA gene are included. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises a Y356P substitution. According to a particular embodiment, the bacterium comprises one or more nucleotide substitutions in the polypeptide encoded in the thrA gene which result in a Y356H substitution. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the Y356H substitution. In a particular embodiment, the bacterium comprises one or more nucleotide substitutions in the polypeptide encoded in the thrA gene that result in a Y356R substitution. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises a Y356R substitution. In a particular embodiment, the bacterium comprises one or more nucleotide substitutions in the polypeptide encoded in the thrA gene that result in a Y356L substitution. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the Y356L substitution.

According to a particular embodiment, the present invention provides a bacterium comprising one or more nucleotide substitutions which result in an amino acid substitution at position S357 in the polypeptide encoded in the thrA gene. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises an amino acid substitution at position S357. According to a specific embodiment, the amino acid substitution is selected from the group consisting of S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M. In another specific embodiment, the amino acid substitutions are selected from the group consisting of S357R, S357V, S357G, S357L, S357Y, S357A, S357N, S357F and S357H. In a more specific embodiment, the amino acid substitutions are selected from the group consisting of S357R, S357A, S357N and S357F. In another more specific embodiment, the amino acid substitutions are selected from the group consisting of S357A and S357F.

In a particular embodiment, the bacterium comprises in the thrA gene one or more nucleotide substitutions that result in a S357R substitution in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the S357R substitution. According to a particular embodiment, the bacterium comprises one or more nucleotide substitutions in the polypeptide encoded in the thrA gene that result in a S357V substitution. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the S357V substitution.

In a particular embodiment, the bacterium comprises one or more nucleotide substitutions that result in a S357P substitution in the polypeptide encoded in the thrA gene. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises a S357P substitution. According to a particular embodiment, the bacterium comprises one or more nucleotide substitutions in the polypeptide encoded in the thrA gene that result in a S357G substitution. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the S357G substitution. According to a particular embodiment, the bacterium comprises one or more nucleotide substitutions in the thrA gene which result in a S357L substitution in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the S357L substitution. According to a particular embodiment, the bacterium comprises one or more nucleotide substitutions in the polypeptide encoded in the thrA gene that result in a S357Y substitution. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the S357Y substitution. In a particular embodiment, the bacterium comprises one or more nucleotide substitutions in the polypeptide encoded in the thrA gene that result in a S357A substitution. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the S357A substitution. According to a particular embodiment, the bacterium comprises one or more nucleotide substitutions in the polypeptide encoded in the thrA gene that result in a S357N substitution. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the S357N substitution. In a particular embodiment, the bacterium comprises in the thrA gene one or more nucleotide substitutions that result in a S357F substitution in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the S357F substitution.

In a particular embodiment, the bacterium comprises in the thrA gene one or more nucleotide substitutions that result in a S357H substitution in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the S357H substitution.

In a particular embodiment, the bacterium comprises in the thrA gene one or more nucleotide substitutions that result in a S357K substitution in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the S357K substitution. In a particular embodiment, the bacterium comprises one or more nucleotide substitutions in the polypeptide encoded in the thrA gene that result in a S357I substitution. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the S357I substitution. In a particular embodiment, the bacterium comprises in the thrA gene one or more nucleotide substitutions that result in a S357M substitution in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the S357M substitution.

According to a particular embodiment, the invention provides a bacterium comprising in the thrA gene one or more nucleotide substitutions which result in an amino acid substitution at position S359 in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises an amino acid substitution at position S359. According to a specific embodiment, the amino acid substitution is selected from the group consisting of S359R, S359G, S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L. According to another specific embodiment, the amino acid substitution is selected from the group consisting of S359R, S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L. In a more specific embodiment, the amino acid substitutions are selected from the group consisting of S359R, S359T, S359P, S359V, S359Q, S359A, S359E and S359L. In another more specific embodiment, the amino acid substitutions are selected from the group consisting of S359R, S359T, S359P, S359Q, S359A, S359E and S359L. In another more specific embodiment, the amino acid substitutions are selected from the group consisting of S359R, S359T, S359Q, S359A and S359E. In another more specific embodiment, the amino acid substitutions are selected from the group consisting of S359R, S359T and S359A. In another more specific embodiment, the amino acid substitutions are selected from the group consisting of S359R and S359A.

In a particular embodiment, the bacterium comprises in the thrA gene one or more nucleotide substitutions that result in a S359R substitution in the encoded polypeptide. The bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein the polypeptide contains a S359R substitution. In a particular embodiment, the bacterium comprises in the thrA gene one or more nucleotide substitutions that result in a S359G substitution in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the S359G substitution.

According to a particular embodiment, the present invention provides a bacterium comprising within the thrA gene one or more nucleotide substitutions which result in a S359M substitution in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the S359M substitution. According to a particular embodiment, one or more nucleotide substitutions that result in S359F substitutions in the encoded polypeptide are included in the thrA gene. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the S359F substitution.

In a particular embodiment, the bacterium comprises in the thrA gene one or more nucleotide substitutions that result in a S359T substitution in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the S359T substitution. In a particular embodiment, the bacterium comprises in the thrA gene one or more nucleotide substitutions that result in a S359P substitution in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the S359P substitution.

In a particular embodiment, the bacterium comprises in the thrA gene one or more nucleotide substitutions that result in an S359V substitution in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the S359V substitution. In a particular embodiment, the bacterium comprises in the thrA gene one or more nucleotide substitutions that result in an S359Q substitution in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the S359Q substitution.

According to a particular embodiment, the bacterium comprises in the thrA gene one or more nucleotide substitutions which result in a S359A substitution in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the S359A substitution. In a particular embodiment, the bacterium comprises in the thrA gene one or more nucleotide substitutions that result in a S359C substitution in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the S359C substitution.

In a particular embodiment, the bacterium comprises in the thrA gene one or more nucleotide substitutions that result in a S359K substitution in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the S359K substitution.

In a particular embodiment, the bacterium produces an S359E substitution in the encoded polypeptide within the thrA gene. Includes one or more nucleotide substitutions. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the S359E substitution.

In a particular embodiment, the bacterium comprises in the thrA gene one or more nucleotide substitutions that result in a S359L substitution in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the S359L substitution.

According to a particular embodiment, the invention provides one or more nucleotide substitutions within the thrA gene that result in amino acid substitutions at position Y356 in the encoded polypeptide, at position S357 in the encoded polypeptide. There is provided a bacterium comprising one or more nucleotide substitutions that result in an amino acid substitution and/or one or more nucleotide substitutions that result in an amino acid substitution at position S359 in the encoded polypeptide; at the position Y356. Is substituted from the group consisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L.
The substitution at position S357 is selected from the group consisting of S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; and the substitution at position S359 is It is selected from the group consisting of S359R, S359G, S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

In a particular embodiment, the invention provides one or more nucleotide substitutions in the thrA gene that result in an amino substitution at position Y356 in the encoded polypeptide and/or position S357 in the encoded polypeptide. Providing a bacterium comprising one or more nucleotide substitutions that result in amino acid substitutions at: Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Selected from the group consisting of Y356F, Y356A, Y356I, Y356P, Y356H, Y356R, Y356L; the substitution at the position of S357 is S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F. It is selected from the group consisting of S357I and S357M.

In a particular embodiment, the invention provides one or more nucleotide substitutions in the thrA gene that result in an amino substitution at position Y356 in the encoded polypeptide and/or position S359 in the encoded polypeptide. Providing a bacterium comprising one or more nucleotide substitutions that result in amino acid substitutions at: Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E. , Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and are selected from the group consisting of Y356L; substituted at and S359 are, S359R, S359G, S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C , S359K, S359E and S359L.

In a particular embodiment, the invention provides one or more nucleotide substitutions that result in an amino substitution at position S357 in the encoded polypeptide, and/or amino acids at position S359 in the encoded polypeptide. There is provided a bacterium comprising a substitution of one or more nucleotides in the thrA gene that results in the substitution; the substitution at the position of S357, wherein the substitution at S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F. , S357H, S357K, S357I and S357M; and the substitution at the position of S359 is S359R, S359G, S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359S, 359K, S359K and S359K, S359S, S359K, S359S and S359K. Is selected from the group consisting of:

According to a particular embodiment, the invention provides one or more nucleotide substitutions that result in an amino substitution at position Y356 in the encoded polypeptide, amino substitutions at position S357 in the encoded polypeptide. There is provided a bacterium comprising in the thrA gene one or more nucleotide substitutions and one or more nucleotide substitutions that result in an amino substitution at position S359 in the encoded polypeptide; at the position of Y356 Substitutions are selected from the group consisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356R and 35 positions; , S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; and the substitution at the position of S359 is S359R, S359S, S359G, S359G, S359G. , S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

In a particular embodiment, the invention provides one or more nucleotide substitutions that result in an amino substitution at position Y356 in the encoded polypeptide and amino acid substitutions at position S357 in the encoded polypeptide. A bacterium comprising a substitution of one or more nucleotides in the thrA gene is provided; wherein the substitution at the Y356 position is Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F. , Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; and the substitution at the position of S357 is S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357S, 357F, S357F, S357F, S357F, S357F, S357K. , S357I and S357M.

In a particular embodiment, the invention provides one or more nucleotide substitutions within the thrA gene that result in an amino substitution at position Y356 in the encoded polypeptide and the amino acid at position S359 in the encoded polypeptide. Providing a bacterium comprising one or more nucleotide substitutions that result in a substitution; the substitution at position Y356 is such that the substitution at Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F. And a substitution at the position of S359 is selected from the group consisting of S359R, S359G, S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L. R.

According to a particular embodiment, the invention provides one or more nucleotide substitutions in the thrA gene that result in an amino substitution at position S357 in the encoded polypeptide, and the position of S359 in the encoded polypeptide. Providing a bacterium comprising one or more nucleotide substitutions that result in an amino acid substitution at S357; , S357K, S357I and S357M; and the substitution at the position of S359 comprises S359R, S359G, S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359E. Selected from the group.

According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 comprising amino acid substitutions at positions Y356, S357 and/or S359; wherein the position of Y356. The substitution at 5 is selected from the group consisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; The substitution is selected from the group consisting of S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; and the substitution at the position of S359 is S359R, S359G, S359G, S359G, S359G, S359G, S359G, S357G. , S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

In a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 which comprises an amino acid substitution at position Y356 and/or S357; The substitution is selected from the group consisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356R and 35 positions; and S. The substitution is selected from the group consisting of S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M.

According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 which comprises an amino acid substitution at position Y356 and/or S359; The substitutions are selected from the group 35 of positions consisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; and S. The substitution is selected from the group consisting of S359R, S359G, S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 which comprises an amino acid substitution at position S357 and/or S359; The substitution is selected from the group consisting of S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; , S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

In a particular embodiment, the bacterium of the present invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 that includes an amino acid substitution at position Y356; wherein the substitution at position Y356 is Y356C. , Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L.

According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 which comprises an amino acid substitution at position S357; wherein the substitution at position S357 is S357R. , S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; , S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 which comprises an amino acid substitution at position S359; wherein the substitution at position S359 is S359R. , S359G, S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, which comprises amino acid substitutions at positions Y356 and S357; wherein the substitution at position Y356 is , Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F and Y356A; , S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M.

According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 which comprises amino acid substitutions at positions Y356 and S359; wherein the substitution at position Y356 is , Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F and Y356A, Y356I, Y356P, Y356H, Y356R and Y356L are selected from the group consisting of: and S359. , S359R, S359G, S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, which comprises amino acid substitutions at positions S357 and S359; wherein the substitution at position S357 is , S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; , S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, which comprises amino acid substitutions at positions Y356, S357 and S359; The substitution is selected from the group consisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R, and Y356L, 35; Is selected from the group consisting of S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; and the substitution at the position of S359 is S359R, S359G, S359. Is selected from the group consisting of S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which the tyrosine is replaced by a cysteine at position 356. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which the tyrosine is replaced by threonine at position 356. In a particular embodiment, the bacterium of the present invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, wherein tyrosine is replaced by valine at position 356. In a particular embodiment, the bacterium of the present invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 in which tyrosine is replaced by serine at position 356. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which at position 356 the tyrosine is replaced by tryptophan. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which the tyrosine at position 356 is replaced by glutamine. In a particular embodiment, the bacterium of the present invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 in which glycine is substituted for tyrosine at position 356. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which the tyrosine at position 356 is replaced by asparagine.

According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which the tyrosine at position 356 is replaced by aspartic acid. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which the tyrosine at position 356 is replaced by glutamic acid. In a particular embodiment, the bacterium of the present invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 in which tyrosine is replaced by phenylalanine at position 356. In a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 in which tyrosine is replaced with alanine at position 356. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which at position 356 tyrosine is replaced by isoleucine. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which the tyrosine at position 356 is replaced by proline. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which the tyrosine at position 356 is replaced by histidine. In a particular embodiment, the bacterium of the present invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 in which tyrosine is replaced by arginine at position 356. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which tyrosine is replaced by leucine at position 356.

According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which at position 357 serine is replaced by arginine. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which at position 357 serine is replaced by valine. In a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 in which at position 357 serine is replaced by proline. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which at position 357 serine is replaced by glycine. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which at position 357 serine is replaced by leucine. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which at position 357 serine is replaced by tyrosine.

According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which at position 357 serine is replaced by alanine. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which at position 357 serine is replaced by asparagine. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which at position 357 serine is replaced by phenylalanine. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which at position 357 serine is replaced by histidine.

According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which at position 357 serine is replaced by lysine. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which at position 357 serine is replaced by isoleucine. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which at position 357 serine is replaced by methionine.

According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which at position 359 serine is replaced by arginine. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which at position 359 serine is replaced by glycine. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which at position 359 serine is replaced by methionine. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which at position 359 serine is replaced by phenylalanine. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which at position 359 serine is replaced by threonine. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which at position 359 serine is replaced by proline. In a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 in which at position 359 serine is replaced by valine.

According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which at position 359 serine is replaced by glutamine. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which at position 359 serine is replaced by alanine. In a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 in which at position 359 serine is replaced by cysteine. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which at position 359 serine is replaced by lysine. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which at position 359 serine is replaced by glutamic acid. According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, in which at position 359 serine is replaced by leucine.

According to a particular embodiment, the bacterium of the invention comprises at least about 90%, for example at least about 93% amino acid sequence according to SEQ ID NO: 11, comprising an amino acid substitution at position Y356, S357 and/or S359, Expressing a polypeptide having an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence homology, wherein the substitution at position Y356 is , Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L are selected from the group consisting of: and S357. , S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; substitution at the position of S359 is S359R, S359G, S359M, S359G, S359M. It is selected from the group consisting of S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

In a particular embodiment, the bacterium of the invention has at least about 90%, for example at least about 93%, at least about at least about 90% of the amino acid sequence set forth in SEQ ID NO: 11, which comprises amino acid substitutions at positions Y356 and/or S357. Expressing a polypeptide having an amino acid sequence having 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence homology, wherein the substitution at the Y356 position is Y356C. , Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; , S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M.

According to a particular embodiment, the bacterium of the invention comprises at least about 90%, for example at least about 93%, at least about 90% amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 11, which comprises an amino acid substitution at position Y356 and/or S359. Expressing a polypeptide having an amino acid sequence having 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence homology, wherein the substitution at the Y356 position is Y356C. , Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; , S359G, S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

In a particular embodiment, the bacterium of the invention has at least about 90%, for example at least about 93%, at least about at least 90% of the amino acid sequence set forth in SEQ ID NO: 11 comprising amino acid substitutions at positions S357 and/or S359. Expressing a polypeptide having an amino acid sequence having 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence homology, wherein the substitution at position S357 is replaced with S357R. , S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; , S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

In a particular embodiment, the bacterium of the invention has at least about 90%, for example at least about 93%, at least about 95%, at least about 95%, at least about 95% amino acid sequence according to SEQ ID NO: 11, which comprises an amino acid substitution at position Y356. Expressing a polypeptide having an amino acid sequence having a sequence homology of about 96%, at least about 97%, at least about 98% or at least about 99%, wherein the substitution at the Y356 position is Y356C, Y356T, Y356V. , Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L.

According to a particular embodiment, the bacterium of the present invention has at least about 90%, for example at least about 93%, at least about 95%, at least about 95%, at least about 95% amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 11, which comprises an amino acid substitution at position S357. Expressing a polypeptide having an amino acid sequence having a sequence homology of about 96%, at least about 97%, at least about 98% or at least about 99%, wherein the substitution at the position of S357 is S357R, S357V, S357P. , S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M.

According to a particular embodiment, the bacterium of the present invention has at least about 90%, for example at least about 93%, at least about 95%, at least about 95%, at least about 95% amino acid sequence according to SEQ ID NO: 11, which comprises an amino acid substitution at position S359. A polypeptide having an amino acid sequence having a sequence homology of about 96%, at least about 97%, at least about 98% or at least about 99% is expressed, wherein the substitution at the position of S359 is S359R, S359G, S359M. , S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

According to a particular embodiment, the bacterium of the invention has at least about 93%, for example at least about 95%, at least about 96%, at least about at least about 93% amino acid sequence according to SEQ ID NO: 11 comprising an amino acid substitution at position Y356. A polypeptide having an amino acid sequence having 97%, at least about 98% or at least about 99% sequence homology is expressed, wherein the substitution at the Y356 position is Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q. , Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L.

According to a particular embodiment, the bacterium of the invention has at least about 93%, for example at least about 95%, at least about 96%, at least about at least about 93% amino acid sequence according to SEQ ID NO: 11 comprising an amino acid substitution at position S357. Express a polypeptide having an amino acid sequence having 97%, at least about 98% or at least about 99% sequence homology, wherein the substitution at the position of S357 is S357R, S357V, S357P, S357G, S357L, S357Y , S357A, S357N, S357F, S357H, S357K, S357I and S357M.

In a particular embodiment, the bacterium of the invention has at least about 93%, for example at least about 95%, at least about 96%, at least about at least about 93% amino acid sequence according to SEQ ID NO: 11 comprising an amino acid substitution at position S359. Expresses a polypeptide having an amino acid sequence having 97%, at least about 98% or at least about 99% sequence homology, wherein the substitution at the position of S359 is S359R, S359G, S359M, S359F, S359T, S359P , S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

In a particular embodiment, the bacterium of the present invention has at least about 95%, for example at least about 96%, at least about 97%, at least about at least about 95% amino acid sequence as set forth in SEQ ID NO: 11 comprising an amino acid substitution at position Y356. A polypeptide having an amino acid sequence having 98% or at least about 99% sequence homology is expressed, wherein the substitution at the Y356 position is Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Selected from the group consisting of Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L.

In a particular embodiment, the bacterium of the present invention has at least about 95%, for example at least about 96%, at least about 97%, at least about at least about 95% amino acid sequence according to SEQ ID NO. Expressing a polypeptide having an amino acid sequence with 98% or at least about 99% sequence homology, wherein the substitution at the position of S357 is S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, Selected from the group consisting of S357F, S357H, S357K, S357I and S357M.

In a particular embodiment, the bacterium of the invention has at least about 95%, for example at least 96%, at least about 97%, at least about 98% amino acid sequence according to SEQ ID NO: 11 comprising an amino acid substitution at position S359. %, or a polypeptide having an amino acid sequence having at least about 99% sequence homology, wherein the substitution at the S359 position is S359R, S359G, S359M, S359F, S359T, S359P, S359V, S359Q, S359A. , S359C, S359K, S359E and S359L.

According to a particular embodiment, the bacterium of the invention comprises at least about 90%, for example at least about 93%, at least about 95% amino acid sequence according to SEQ ID NO: 11 comprising amino acid substitutions at positions Y356, S357 and S359. %, at least about 96%, at least about 97%, at least about 98% or at least about 99%, a polypeptide having an amino acid sequence having sequence homology is expressed, wherein the substitution at the position of Y356 is Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L are selected; the substitution at position 7 at S357S is 7; Selected from the group consisting of S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; It is selected from the group consisting of S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

In a particular embodiment, the bacterium of the invention comprises at least about 90%, such as at least about 93%, at least about 95% amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 11, which comprises amino acid substitutions at positions Y356 and S357, Expressing a polypeptide having an amino acid sequence having at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence homology, wherein the substitution at position Y356 is Y356C, Y356T, Selected from the group consisting of Y356V, Y356S, Y356W, Y356Q, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R, and Y356L; It is selected from the group consisting of S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M.

In a particular embodiment, the bacterium of the invention has at least about 90%, for example at least about 93%, at least about 95% amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 11, which comprises amino acid substitutions at positions Y356 and S359. Expressing a polypeptide having an amino acid sequence having at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence homology, wherein the substitution at position Y356 is Y356C, Y356T, Selected from the group consisting of Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R, Y356L; It is selected from the group consisting of S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

According to a particular embodiment, the bacterium of the invention comprises at least about 90%, such as at least about 93%, at least about 95% amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 11, which comprises amino acid substitutions at positions S357 and S359. Expressing a polypeptide having an amino acid sequence having at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence homology, wherein the substitution at position Y356 is Y356C, Y356T, Selected from the group consisting of Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; substitutions S35, S35S, S35R at positions S357, S357R. Selected from the group consisting of S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; the substitution at the position of S359 is S359R, S359G, S359M, S359F, S359S, S359S, S359P. Selected from the group consisting of S359A, S359C, S359K, S359E and S359L.

According to a particular embodiment, the bacterium of the invention expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, which comprises an amino acid substitution at position Y356, S357 and/or S359, wherein the position at Y356. The substitution at 5 is selected from the group consisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; The substitution is selected from the group consisting of S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; and the substitution at the position of S359 is S359R, S359G, S359G, S359G, S359G, S359G, S359G, S357G. , S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L; wherein one or more, for example about 1 to about 50, about 1 to about 40. About 1, about 35, about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 1 to about 5 , Or about 1 to about 3 additional amino acid residues are substituted, deleted and/or inserted.

The bacterium may express a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 that includes amino acid substitutions at positions Y356, S357 and/or S359, wherein the substitution at position Y356 is Y356C, Y356T. , Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; the substitution at position S357 is S35V, S35S, S357R. S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; , S359Q, S359A, S359C, S359K, S359E and S359L; wherein about 1 to about 5, for example about 1 to about 3 additional amino acid residues are substituted, deleted and / Or inserted.

According to a particular embodiment, the invention provides for one or more nucleotide substitutions in the encoded polypeptide that result in one or more amino acid substitutions selected from the group consisting of Y356C, S357R and S359R. Providing bacteria contained within. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide is one or more (eg 2 or more) selected from the group consisting of Y356C, S357R and S359R. Or 3) amino acid substitutions. In a particular embodiment, the bacterium of the invention comprises one or more nucleotide substitutions in the thrA gene, resulting in one selected from the group consisting of Y356C and S357R in the encoded polypeptide. Alternatively, a bacterium that produces multiple (eg, 2) amino acid substitutions is provided. Therefore, the bacterium of the present invention may express the polypeptide encoded by the thrA gene, wherein said polypeptide is one selected from the group consisting of Y356C and S357R in the encoded polypeptide. Alternatively, it includes multiple (eg, 2) amino acid substitutions. In a particular embodiment, the bacterium of the invention comprises one or more (eg 2) amino acid substitutions in the encoded polypeptide that result in one or more (eg 2) amino acid substitutions selected from the group consisting of Y356C and S359R. Nucleotide substitutions within the thrA gene. Thus, the bacterium of the present invention may express a polypeptide encoded by thrA, wherein said polypeptide is one of the groups selected from the group consisting of Y356C and S359R in the encoded polypeptide. It includes multiple (eg, 2) amino acid substitutions. In a particular embodiment, the bacterium of the invention comprises one or more (eg 2) amino acid substitutions in the encoded polypeptide that result in one or more (eg 2) amino acid substitutions selected from the group consisting of S357R and S359R. Nucleotide substitutions within the thrA gene. Therefore, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein the polypeptide is one or more selected from the group consisting of S357R and S359R in the encoded polypeptide. It includes multiple (eg, 2) amino acid substitutions.

In a particular embodiment, the bacterium comprises in the thrA gene one or more nucleotide substitutions that result in a Y356C substitution in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises a Y356C substitution. In a particular embodiment, the bacterium comprises in the thrA gene one or more nucleotide substitutions that result in a S357R substitution in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the S357R substitution. In a particular embodiment, the bacterium comprises in the thrA gene one or more nucleotide substitutions that result in a S359R substitution in the encoded polypeptide. Thus, the bacterium of the present invention may express a polypeptide encoded by the thrA gene, wherein said polypeptide comprises the S359R substitution.

Thus, the bacterium of the present invention may express aspartate kinase I/homoserine dehydrogenase I (ThrA) having one or more amino acid substitutions selected from the group consisting of Y356C, S357R and S359R. More specifically, the bacterium of the present invention expresses a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 11 containing one or more amino acid substitutions selected from the group consisting of Y356C, S357R and S359R. Good.

In a particular embodiment, the bacterium expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 in which tyrosine is replaced with cysteine at position 356. In a particular embodiment, the bacterium expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 in which at position 357 serine is replaced by arginine. In a particular embodiment, the bacterium expresses a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 in which at position 359 serine is replaced by arginine.

In a particular embodiment, the bacterium has at least about 90%, at least about 93% of the amino acid sequence set forth in SEQ ID NO: 11 that comprises one or more amino acid substitutions selected from the group consisting of Y356C, S357R and S359R. Expresses a polypeptide having an amino acid sequence having sequence homology of at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

In a particular embodiment, the bacterium has at least about 90%, at least about 93%, at least about 95%, at least about 96 amino acid sequence according to SEQ ID NO: 11 in which the tyrosine is replaced with a cysteine at position 356. Express a polypeptide having an amino acid sequence having a sequence homology of at least about 97%, at least about 97%, at least about 98%, or at least about 99%.

In a specific embodiment, the bacterium is at least about 95%, for example at least about 96%, at least about 97%, to the amino acid sequence set forth in SEQ ID NO: 11, wherein tyrosine is replaced with cysteine at position 356. Express a polypeptide having an amino acid sequence that has at least about 98%, or at least about 99% sequence homology.

In certain embodiments, the bacterium comprises at least about 90%, at least about 93%, at least about 95%, at least about 96 amino acid sequence according to SEQ ID NO: 11, wherein serine is substituted for arginine at position 357. Express a polypeptide having an amino acid sequence having a sequence homology of at least about 97%, at least about 97%, at least about 98%, or at least about 99%. In a specific embodiment, the bacterium comprises at least about 95%, at least about 96%, at least about 97%, at least about 95% of the amino acid sequence set forth in SEQ ID NO: 11, wherein serine is substituted for arginine at position 357. Express polypeptides having an amino acid sequence with 98%, or at least about 99% sequence homology.

In a particular embodiment, the bacterium has at least about 90%, at least about 93%, at least about 95%, at least about 96 amino acid sequence according to SEQ ID NO: 11, wherein serine is replaced by arginine at position 359. Express a polypeptide having an amino acid sequence having a sequence homology of at least about 97%, at least about 97%, at least about 98%, or at least about 99%.

In a specific embodiment, the bacterium has at least about 95%, at least about 96%, at least about 97%, at least about 95% of the amino acid sequence set forth in SEQ ID NO: 11 in which serine is replaced by arginine at position 359. Express polypeptides having an amino acid sequence with 98%, or at least about 99% sequence homology.

In a particular embodiment, the bacterium expresses a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 11, which comprises one or more amino acid substitutions selected from the group consisting of Y356C, S357R and S359R, wherein One or more, for example about 1 to about 50, about 1 to about 40, about 1 to about 35, about 1 to about 30, about 1 to about 25, about 1 to about 20 , About 1 to about 15, about 1 to about 10, about 1 to about 5, or about 1 to about 3 additional amino acid residues have been substituted, deleted and/or inserted. The bacterium comprises one or more amino acid substitutions selected from the group consisting of Y356C, S357R and S359R, wherein about 1 to about 5, or about 1 to about 3 additional amino acid residues are: A polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 that has been substituted, deleted and/or inserted may be expressed.

The ThrA polypeptide mutants described above may be (over)expressed by the bacterium using an exogenous nucleic acid molecule such as an expression vector that has been introduced into the bacterium. Thus, in a particular embodiment, the bacterium of the invention comprises an exogenous nucleic acid molecule comprising a nucleotide sequence encoding an aspartate kinase I/homoserine dehydrogenase I (ThrA) polypeptide mutant as described above. Including.

For example, the bacterium of the present invention may be characterized by an exogenous nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 that includes amino acid substitutions at positions Y356, S357 and/or S359, such as expression. It may include a vector. Thus, according to a particular embodiment, the bacterium of the invention comprises a nucleotide sequence encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 which comprises an amino acid substitution at position Y356, S357 and/or S359. Exogenous nucleic acid molecule, wherein the substitution at position Y356 is Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, And a substitution at the position of S357 is selected from the group consisting of S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; The substitution at the position is selected from the group consisting of S359R, S359G, S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

In a particular embodiment, the bacterium of the invention has at least about 90%, for example at least about 93%, at least about 93% amino acid sequence in the amino acid sequence set forth in SEQ ID NO: 11 comprising amino acid substitutions at positions Y356, S357 and/or S359. An exogenous nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having an amino acid sequence having a sequence homology of about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. Including. According to a particular embodiment, the bacterium of the invention has at least about 90%, for example at least about 93%, at least about at least about 90% amino acid sequence according to SEQ ID NO: 11 comprising amino acid substitutions at positions Y356, S357 and/or S359. An exogenous nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having an amino acid sequence having 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence homology. At that time, the substitution at the position of Y356 is Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R, and Y356R and Y356R and Y356R. A substitution at position S357 is selected from the group consisting of S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; and a substitution at position S359. Is selected from the group consisting of S359R, S359G, S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

In a particular embodiment, the bacterium of the invention has at least about 93%, at least about 95%, at least about 96% amino acid sequence as set forth in SEQ ID NO:11 comprising amino acid substitutions at positions Y356, S357 and/or S359. %, at least about 97%, at least about 98%, or at least about 99% of an exogenous nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide having an amino acid sequence having sequence homology. In a particular embodiment, the bacterium of the invention has at least about 93%, at least about 95%, at least about 96% amino acid sequence as set forth in SEQ ID NO:11 comprising amino acid substitutions at positions Y356, S357 and/or S359. %, at least about 97%, at least about 98%, or at least about 99% of an exogenous nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide having an amino acid sequence having a sequence homology, wherein Y356 The substitution at position is selected from the group consisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; Is selected from the group consisting of S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; and the substitution at the position of S359 is S359R, S359G,. It is selected from the group consisting of S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

In a particular embodiment, the bacterium of the invention has at least about 95%, at least about 96%, at least about 97% amino acid sequence as set forth in SEQ ID NO:11 comprising amino acid substitutions at positions Y356, S357 and/or S359. %, at least about 98%, or at least about 99% sequence homology with an exogenous nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide having an amino acid sequence. In a particular embodiment, the bacterium of the invention has at least about 95%, at least about 96%, at least about 97% amino acid sequence as set forth in SEQ ID NO:11 comprising amino acid substitutions at positions Y356, S357 and/or S359. %, at least about 98%, or at least about 99% sequence homology, comprising an exogenous nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having an amino acid sequence, wherein the substitution at position Y356 is , Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356R, and 7 are selected from the group S; , S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; , S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

In this regard, the invention further provides (isolated) nucleic acid molecules, such as expression vectors, containing a nucleotide sequence encoding a ThrA mutant as described above. Such nucleic acid may be introduced into the bacterium of the present invention. In a particular embodiment, the nucleic acid molecule is at least about 90%, such as at least about 93%, at least about 95% amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 11, which comprises amino acid substitutions at positions Y356, S357 and/or S359. %, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence homology, comprising a nucleotide sequence encoding a polypeptide having an amino acid sequence.

In a particular embodiment, the nucleic acid molecule is at least about 90%, such as at least about 93%, at least about 95% amino acid sequence according to SEQ ID NO: 11 comprising amino acid substitutions at positions Y356, S357 and/or S359. %, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, a nucleotide sequence encoding a polypeptide having an amino acid sequence having sequence homology at the position of Y356. Substitutions are selected from the group consisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356R and 35 positions; , S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; and the substitution at the position of S359 is S359R, S359S, S359G, S359G, S359G. , S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L. In a particular embodiment, the nucleic acid molecule comprises a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 that includes amino acid substitutions at positions Y356, S357 and/or S359. In a particular embodiment, the nucleic acid molecule comprises a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 that includes amino acid substitutions at positions Y356, S357 and/or S359, wherein Y356 Substitutions at positions Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; The substitution at is selected from the group consisting of S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; and the substitution at the position of S359 is S359R, S359G. , S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

The (isolated) nucleic acid molecule may be an exogenous nucleic acid as detailed above. The (isolated) nucleic acid molecule may contain suitable regulatory elements such as a promoter that functions in bacterial cells and causes the production of mRNA molecules, which is operative for the nucleotide sequence encoding the polypeptide. Connected to. Further details on suitable regulatory elements are provided below for "exogenous" nucleic acid molecules and apply mutatis mutandis.

The present invention provides a bacterium containing within the Irp gene one or more nucleotide substitutions that result in the amino acid substitution D143G in the encoded polypeptide. For example, further information regarding the E. coli Irp is available at EcoCyc (www.biocyc.org) under accession number EG10547. According to a particular embodiment, the bacterium of the invention expresses the polypeptide encoded by the Irp gene, wherein D is replaced by G at position 143 in said polypeptide. A representative amino acid sequence for the polypeptide encoded by the Irp gene is set forth in SEQ ID NO:12. According to a specific embodiment, the bacterium of the invention comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 12, or the amino acid sequence set forth in SEQ ID NO: 12 (wherein the amino acid sequence is at position 143 D Are substituted for G) with at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence homology. Express the polypeptide.

According to a particular embodiment, the invention provides a bacterium comprising one or more substitutions in the Irp gene that result in one or more amino acid substitutions that increase resistance to L-serine. More specifically, there is provided a bacterium comprising within the Irp gene one or more nucleotide substitutions that result in an amino acid substitution at position D143 in the encoded polypeptide. According to a particular embodiment, the bacterium of the invention comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 12, or the amino acid sequence set forth in SEQ ID NO: 12 (wherein said amino acid sequence is at position 143 where D is At least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. Express a polypeptide having. Advantageously, the one or more amino acid substitutions are non-conservative substitutions.

The invention also provides a bacterium comprising within the rho gene one or more nucleotide substitutions that result in the amino acid substitution R87L in the encoded polypeptide. For example, information regarding E. coli is available on EcoCyc (www.biocyc.org) under accession number EG10845. According to a particular embodiment, the bacterium of the invention expresses the polypeptide encoded by the rho gene, wherein R is replaced by L at position 87 in said polypeptide.

A representative amino acid sequence for the polypeptide encoded by the rho gene is set forth in SEQ ID NO:13. According to a suitable embodiment, the bacterium of the present invention comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 13, or the amino acid sequence set forth in SEQ ID NO: 13, wherein R is at position 87 in the amino acid sequence. Substituted with L) has a sequence homology of at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. Express the peptide.

According to a particular embodiment, the present invention provides a bacterium comprising in the rho gene one or more nucleotide substitutions that result in one or more amino acid substitutions that increase resistance to L-serine. More specifically, there is provided a bacterium comprising one or more nucleotide substitutions that result in an amino acid substitution in the R87 position in the encoded polypeptide. According to a particular embodiment, the bacterium of the invention comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 13, or the amino acid sequence set forth in SEQ ID NO: 13 (wherein the amino acid sequence is R at position 87). Are substituted with other amino acids) at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence homology. Expressing a polypeptide having Advantageously, the one or more amino acid substitutions are non-conservative substitutions.

The invention also provides a bacterium comprising within the eno gene one or more nucleotide substitutions that result in the amino acid substitution V164L in the encoded polypeptide. For example, additional information regarding E. coli is available at EcoCyc (www.biocyc.org) under accession number EG10258. According to a particular embodiment, the bacterium of the invention expresses a polypeptide encoded by the eno gene, wherein V is replaced by L at position 164 in said polypeptide. A representative amino acid sequence for the polypeptide encoded by the eno gene is set forth in SEQ ID NO:14. According to a particular embodiment, the bacterium of the invention comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:14, or the amino acid sequence set forth in SEQ ID NO:14, wherein said amino acid sequence is Substituted with L) has a sequence homology of at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. Express the peptide.

According to a particular embodiment, the present invention provides a bacterium comprising one or more nucleotide substitutions in the eno gene that result in one or more amino acid substitutions that increase resistance to L-serine. More specifically, there is provided a bacterium comprising in the eno gene one or more nucleotide substitutions that result in an amino acid substitution at position V164 in the encoded polypeptide. According to a particular embodiment, the bacterium of the invention comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:14, or the amino acid sequence set forth in SEQ ID NO:14, wherein said amino acid sequence is At least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. Express a polypeptide having. Advantageously, the one or more amino acid substitutions are non-conservative substitutions.

The invention also provides a bacterium that comprises within the argP gene one or more nucleotide substitutions that result in the amino acid substitution V164L in the encoded polypeptide. For example, additional information regarding E. coli argP is available at EcoCyc (www.biocyc.org) under accession number EG10490. According to a particular embodiment, the bacterium of the invention expresses a polypeptide encoded by the argP gene, wherein Q is replaced by K at position 132 in said polypeptide. A representative amino acid sequence for the polypeptide encoded by the argP gene is set forth in SEQ ID NO:15. According to a particular embodiment, the bacterium of the invention comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:15, or the amino acid sequence set forth in SEQ ID NO:15, wherein said amino acid sequence is at position 132 with a Q Substituted with K) of at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence homology. Express the peptide.

According to a particular embodiment, the invention provides a bacterium comprising in the argP gene one or more nucleotide substitutions that result in one or more amino acid substitutions that increase resistance to L-serine. More specifically, a bacterium is provided that contains within the argP gene one or more nucleotide substitutions that result in an amino acid substitution at position Q132 in the encoded polypeptide. According to a particular embodiment, the bacterium of the invention comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:15, or the amino acid sequence set forth in SEQ ID NO:15, wherein said amino acid sequence is at position 132 with a Q At least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. Express a polypeptide having. Advantageously, the one or more amino acid substitutions are non-conservative substitutions.

The invention also provides a bacterium comprising in the tufA gene one or more nucleotide substitutions that result in the amino acid substitution G19V in the encoded polypeptide. For example, further information regarding E. coli tufA is available at EcoCyc (www.biocyc.org) under accession number EG11036. According to a particular embodiment, the bacterium of the invention expresses a polypeptide encoded by the tufA gene, wherein G is replaced by V at position 19 in said polypeptide. A representative amino acid sequence for the polypeptide encoded by the tufA gene is set forth in SEQ ID NO:16. According to a particular embodiment, the bacterium of the invention comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:16, or the amino acid sequence set forth in SEQ ID NO:16, wherein said amino acid sequence is at position 19 with G Substituted with V) at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence homology. Express the peptide.

According to a particular embodiment, the invention provides a bacterium comprising in the tufA gene one or more nucleotide substitutions that result in one or more amino acid substitutions that increase resistance to L-serine. More specifically, there is provided a bacterium comprising in the tufA gene one or more nucleotide substitutions that result in an amino acid substitution at position G19 in the encoded polypeptide. In a particular embodiment, the bacterium of the invention comprises at least about 90%, at least about 93%, at least about 95% of the polypeptide having the amino acid sequence set forth in SEQ ID NO:16, or the amino acid sequence set forth in SEQ ID NO:16. %, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence homology, wherein the amino acid sequence is at position 19 with G replaced by another amino acid. Are expressed). Advantageously, the one or more amino acid substitutions are non-conservative substitutions.

The invention also provides a bacterium comprising within the cycA gene one or more nucleotide substitutions that result in the amino acid substitution I220V in the encoded polypeptide. For example, additional information regarding E. coli cycA is available at EcoCyc (www.biocyc.org) under accession number EG12504. According to a particular embodiment, the bacterium of the invention expresses the polypeptide encoded by the cycA gene, wherein I is replaced by V at position 220 in said polypeptide. A representative amino acid sequence for the polypeptide encoded by the cycA gene is set forth in SEQ ID NO:17. In a particular embodiment, the bacterium of the invention comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:17, or at least about 90%, at least about 93%, at least about 95% amino acid sequence set forth in SEQ ID NO:17. %, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence homology, wherein the amino acid sequence is substituted for I at position 220 by V. ) Is expressed.

According to a particular embodiment, the invention provides a bacterium comprising in the cycA gene one or more nucleotide substitutions that result in one or more amino acid substitutions that increase resistance to L-serine. More specifically, there is provided a bacterium containing in the cycA gene one or more nucleotide substitutions that result in an amino acid substitution at position I220 in the encoded polypeptide. According to a particular embodiment, the bacterium of the invention comprises a polypeptide having the amino acid sequence as set forth in SEQ ID NO: 17, wherein said amino acid sequence is substituted at position 220 with I for another amino acid, Or at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence homology to the amino acid sequence set forth in SEQ ID NO:17. Express a polypeptide having. Advantageously, the one or more amino acid substitutions are non-conservative substitutions.

The invention also provides a bacterium comprising within the rpe gene one or more nucleotide substitutions that result in the amino acid substitution I202T in the encoded polypeptide. For example, additional information regarding E. coli rpe is available at EcoCyc (www.biocyc.org) under accession number M004. According to a particular embodiment, the bacterium of the invention expresses the polypeptide encoded by the rpe gene, wherein I is replaced by T at position 202 in said polypeptide. A representative amino acid sequence for the polypeptide encoded by the rpe gene is set forth in SEQ ID NO:18. According to a particular embodiment, the bacterium of the invention comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:18, or the amino acid sequence set forth in SEQ ID NO:18, wherein said amino acid sequence is at position 202 at position I Substituted with T) has a sequence homology of at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. Express the peptide.

According to a particular embodiment, the invention provides a bacterium comprising in the rpe gene one or more nucleotide substitutions that result in one or more amino acid substitutions that increase resistance to L-serine. More specifically, a bacterium is provided that contains within the rpe gene one or more nucleotide substitutions that result in an amino acid substitution at position 202I in the encoded polypeptide. According to a particular embodiment, the bacterium of the invention comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:18, or the amino acid sequence set forth in SEQ ID NO:18, wherein said amino acid sequence is at position 202 at position I At least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. Express a polypeptide having. Advantageously, the one or more amino acid substitutions are non-conservative substitutions.

The invention also provides a bacterium comprising within the yojl gene one or more nucleotide substitutions that result in the amino acid substitution D334H in the encoded polypeptide. For example, further information regarding E. coli yojl is available at EcoCyc (www.biocyc.org) under accession number EG12070. According to a particular embodiment, the bacterium of the invention expresses a polypeptide encoded by the yojl gene, wherein D is replaced by H at position 334 in said polypeptide. A representative amino acid sequence for the polypeptide encoded by the yojl gene is set forth in SEQ ID NO:19. According to a particular embodiment, the bacterium of the invention comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 19 or the amino acid sequence set forth in SEQ ID NO: 19 wherein said amino acid sequence is at position 334 and D is Substituted with H) has a sequence homology of at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. Express the peptide.

According to a particular embodiment, the invention provides a bacterium comprising in the yojl gene one or more nucleotide substitutions that result in one or more amino acid substitutions that increase resistance to L-serine. More specifically, there is provided a bacterium comprising in the yojl gene one or more nucleotide substitutions that result in an amino acid substitution at position D334 in the encoded polypeptide. According to a particular embodiment, the bacterium of the invention comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 19 or the amino acid sequence set forth in SEQ ID NO: 19 wherein said amino acid sequence is at position 334 and D is At least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. Express a polypeptide having. Advantageously, the one or more amino acid substitutions are non-conservative substitutions.

The invention also provides a bacterium comprising within the hyaF gene one or more nucleotide substitutions that result in the amino acid substitution V120G in the encoded polypeptide. For example, further information regarding E. coli hyaF is available at EcoCyc (www.biocyc.org) under accession number EG10473. In a particular embodiment, the bacterium of the invention expresses a polypeptide encoded by the hyaF gene, wherein V is replaced by G at position 120 in said polypeptide. A representative amino acid sequence for the polypeptide encoded by the hyaF gene is set forth in SEQ ID NO:20. According to a particular embodiment, the bacterium of the invention comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:20, or the amino acid sequence set forth in SEQ ID NO:20, wherein said amino acid sequence is Substituted for G) with at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence homology. Express the peptide.

According to a particular embodiment, the present invention provides a bacterium comprising in the hyaF gene one or more nucleotide substitutions that result in one or more amino acid substitutions that increase resistance to L-serine. More specifically, there is provided a bacterium comprising in the hyaF gene one or more nucleotide substitutions that result in an amino acid substitution at position V120 in the encoded polypeptide.

According to a particular embodiment, the bacterium of the invention comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:20, or the amino acid sequence set forth in SEQ ID NO:20, wherein said amino acid sequence is At least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. Express a polypeptide having. Advantageously, the one or more amino acid substitutions are non-conservative substitutions.

The invention also provides a bacterium that comprises in the pykF gene one or more nucleotide substitutions that result in the amino acid substitution E250 ^* in the encoded polypeptide, where ^* indicates a stop codon. Alternatively, the pykF gene may contain one or more nucleotide substitutions at a position upstream of position 250 resulting in termination of the encoded polypeptide. For example, additional information regarding E. coli pykF is available at EcoCyc (www.biocyc.org) under accession number EG10804. According to a particular embodiment, the bacterium of the invention expresses a polypeptide encoded by the pykF gene, wherein said polypeptide terminates after position 249 or at any position upstream thereof. A representative amino acid sequence for the polypeptide encoded by the pykF gene is set forth in SEQ ID NO:21. In a particular embodiment, the bacterium of the invention has a polypeptide having the amino acid sequence set forth in SEQ ID NO:22, or at least about 90%, at least about 93%, at least about 95% amino acid sequence set forth in SEQ ID NO:22. %, at least about 96%, at least about 97%, at least about 98%, or at least about 99% polypeptide with sequence homology.

According to another particular embodiment, the bacterium of the invention is further modified to attenuate the expression of the pykF gene (eg by inactivating the gene). Attenuation of gene expression and more specifically inactivation can be achieved as previously described herein. For example, Lambda Red mediated gene exchange may be used to inactivate gene expression.

The invention also provides a bacterium comprising in the malT gene one or more nucleotide substitutions that result in the amino acid substitution Q420 ^{* in the} encoded polypeptide, where ^* indicates a stop codon. Alternatively, the malT gene may contain one or more nucleotide substitutions that result in termination at a position upstream of position 420 of the encoded polypeptide. For example, further information regarding E. coli malT is available at EcoCyc (www.biocyc.org) under accession number EG10562. According to a particular embodiment, the bacterium of the invention expresses a polypeptide encoded by the malT gene, wherein said polypeptide terminates after position 419 or at any position upstream thereof. A representative amino acid sequence for the polypeptide encoded by the malT gene is set forth in SEQ ID NO:23. In a particular embodiment, the bacterium of the invention has a polypeptide having the amino acid sequence set forth in SEQ ID NO:24, or at least about 90%, at least about 93%, at least about 95% amino acid sequence set forth in SEQ ID NO:24. %, at least about 96%, at least about 97%, at least about 98%, or at least about 99% polypeptide with sequence homology.

According to another particular embodiment, the bacterium of the invention is further modified to attenuate the expression of the malT gene (eg by inactivating the gene). Attenuation of gene expression and more specifically inactivation can now be achieved as described above. For example, Lambda Red mediated gene exchange may be used to inactivate gene expression.

The invention also provides a bacterium comprising in the rpoB gene one or more nucleotide substitutions that result in the amino acid substitution P520L in the encoded polypeptide. For example, further information regarding E. coli rpoB is available at EcoCyc (www.biocyc.org) under accession number EG10894. According to a particular embodiment, the bacterium of the invention expresses a polypeptide encoded by the rpoB gene, wherein P is replaced by L at position 520 in said polypeptide. A representative amino acid sequence for the polypeptide encoded by the rpoB gene is set forth in SEQ ID NO:25. According to a particular embodiment, the bacterium of the invention comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:25, or the amino acid sequence set forth in SEQ ID NO:25, wherein P at position 520 in said amino acid sequence is Substituted with L) has a sequence homology of at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. Express the peptide.

According to a particular embodiment, the invention provides a bacterium comprising in the rpoB gene one or more nucleotide substitutions that result in one or more amino acid substitutions that increase resistance to L-serine. More specifically, there is provided a bacterium containing in the rpoB gene one or more nucleotide substitutions that result in an amino acid substitution at position P520 in the encoded polypeptide.

According to a particular embodiment, the bacterium of the invention comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:25, or the amino acid sequence set forth in SEQ ID NO:25, wherein said amino acid sequence is at position 520 and P is another. At least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. Express the polypeptide. Advantageously, the one or more amino acid substitutions are non-conservative substitutions.

The present invention also provides a bacterium containing in the fumB gene one or more nucleotide substitutions that result in the amino acid substitution T218P in the encoded polypeptide. For example, further information regarding Escherichia coli fumB is available at EcoCyc (www.biocyc.org) under accession number EG10357. According to a particular embodiment, the bacterium of the invention expresses a polypeptide encoded by the fumB gene, wherein T is replaced by P at position 218 in said polypeptide. A representative amino acid sequence for the polypeptide encoded by the fumB gene is set forth in SEQ ID NO:26. According to a particular embodiment, the bacterium of the invention comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:26, or the amino acid sequence set forth in SEQ ID NO:26, wherein T at position 218 in said amino acid sequence. Are substituted for P) with at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence homology. Express the polypeptide. Advantageously, the one or more amino acid substitutions are non-conservative substitutions.

According to a particular embodiment, the invention provides a bacterium comprising in the fumB gene one or more nucleotide substitutions that result in one or more amino acid substitutions that increase resistance to L-serine. More specifically, there is provided a bacterium comprising one or more nucleotide substitutions that result in an amino acid substitution at position T218 in a polypeptide encoded within the fumB gene. In a particular embodiment, the bacterium of the invention comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:26, or the amino acid sequence set forth in SEQ ID NO:26, wherein said amino acid sequence is at position 218 with a T Substituted with P) at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence homology. Express the peptide. Advantageously, the one or more amino acid substitutions are non-conservative substitutions.

The invention also provides a bacterium comprising in the gshA gene one or more nucleotide substitutions that result in the amino acid substitution A178V in the encoded polypeptide. For example, additional information regarding E. coli gshA is available at EcoCyc (www.biocyc.org) under accession number EG10418. According to a particular embodiment, the bacterium of the invention expresses the polypeptide encoded by the gshA gene, wherein A is replaced by V at position 178 in said polypeptide. A representative amino acid sequence for the polypeptide encoded by the gshA gene is set forth in SEQ ID NO:27. According to a particular embodiment, the bacterium of the invention comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:27, or the amino acid sequence set forth in SEQ ID NO:27, wherein A at position 178 in said amino acid sequence. Are substituted for V) with at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence homology. Express the polypeptide.

According to a particular embodiment, the present invention provides a bacterium comprising one or more substitutions in the gshA gene that result in one or more amino acid substitutions that increase resistance to L-serine. More specifically, a bacterium is provided that contains within the gshA gene one or more nucleotide substitutions that result in an amino acid substitution at position A178 in the encoded polypeptide. According to a particular embodiment, the bacterium of the invention comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:27, or the amino acid sequence set forth in SEQ ID NO:27, wherein A at position 178 in said amino acid sequence. Are substituted with other amino acids) at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence homology. Expressing a polypeptide having Advantageously, the one or more amino acid substitutions are non-conservative substitutions.

The invention also provides a bacterium comprising in the lamB gene one or more nucleotide substitutions that result in the amino acid substitution Q112 ^{* in the} encoded polypeptide, where ^* indicates a stop codon. Alternatively, the lamB gene may contain one or more nucleotide substitutions that result in termination of the encoded polypeptide at position upstream of position 112. For example, further information regarding E. coli lamB is available at EcoCyc (www.biocyc.org) under accession number EG10528. According to a particular embodiment, the bacterium of the invention expresses a polypeptide encoded by the lamB gene, wherein said polypeptide terminates after position 111 or at any position upstream thereof. .. A representative amino acid sequence for the polypeptide encoded by the lamB gene is set forth in SEQ ID NO:28. In a particular embodiment, the bacterium of the invention has a polypeptide having the amino acid sequence set forth in SEQ ID NO:29, or at least about 90%, at least about 93%, at least about 95% amino acid sequence set forth in SEQ ID NO:29. %, at least about 96%, at least about 97%, at least about 98%, or at least about 99% polypeptide with sequence homology.

According to another particular embodiment, the bacterium of the present invention is further denatured to attenuate the expression of the lamB gene (eg by inactivating the gene). Attenuation of gene expression and more specifically inactivation can be achieved as previously described herein. For example, Lambda Red mediated gene exchange may be used to inactivate gene expression.

The bacterium of the invention comprises one or more, for example two or more, three or more, four or more, or five or more genes as defined above. It may contain a mutation.

For example, the bacterium may contain one or more (eg two or more) genetic mutations selected from the group consisting of:
One or more nucleotide substitutions in the Irp gene resulting in an amino acid substitution at position D143 in the encoded polypeptide,
one or more nucleotide substitutions in the rho gene that result in an amino acid substitution at position R87 in the encoded polypeptide,
Within the eno gene, one or more nucleotide substitutions that result in an amino acid substitution at position V164 in the encoded polypeptide, and in the argP gene, an amino acid substitution at position V164 in the encoded polypeptide 1. One or more nucleotide substitutions.

For example, the bacterium may contain one or more (eg two or more) genetic mutations selected from the group consisting of:
One or more nucleotide substitutions in the Irp gene that result in the amino acid substitution D143G in the encoded polypeptide,
one or more nucleotide substitutions in the rho gene that result in the amino acid substitution R87L in the encoded polypeptide,
One or more nucleotide substitutions in the eno gene that result in amino acid substitution V164L in the encoded polypeptide, and one or more nucleotide substitutions in the argP gene that result in amino acid substitution V164L in the encoded polypeptide.

According to a particular embodiment, the bacterium of the invention comprises one or more nucleotide substitutions in the thrA gene that result in an amino acid substitution at position Y356 in the encoded polypeptide, said substitutions being Y356C, Y356T, Selected from the group consisting of Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; the amino acid substitution at position S357 in the encoded polypeptide. Including one or more nucleotide substitutions that occur, said substitutions being selected from the group consisting of S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; and/or Or one or more nucleotide substitutions that result in amino acid substitutions at position S359 in the encoded polypeptide, said substitutions being S359R, S359G, S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C, Selected from the group consisting of S359K, S359E and S359L; and:
One or more nucleotide substitutions in the Irp gene that result in an amino acid substitution at position D143 in the encoded polypeptide (eg, D143G),
one or more nucleotide substitutions in the rho gene that result in an amino acid substitution at position R87 in the encoded polypeptide (eg, R87L),
Within the eno gene, one or more nucleotide substitutions that result in an amino acid substitution at position V164 in the encoded polypeptide (eg, V164L), and within the argP gene at position V164 in the encoded polypeptide. Of one or more (eg, 2 or more) genetic mutations selected from one or more nucleotide substitutions that result in an amino acid substitution of (eg, V164L).

In a particular embodiment, the bacterium of the invention comprises one or more nucleotide substitutions that result in one or more amino acid substitutions in the encoded polypeptide selected from the group consisting of Y356C, S357R and S359R. contained within the thrA gene; as well as:
One or more nucleotide substitutions in the Irp gene that result in the amino acid substitution D143G in the encoded polypeptide, or one or more nucleotide substitutions in the rho gene that result in the amino acid substitution R87L in the encoded polypeptide. ,
One or more nucleotide substitutions in the eno gene that result in amino acid substitution V164L in the encoded polypeptide, and one or more nucleotide substitutions in the argP gene that result in amino acid substitution V164L in the encoded polypeptide. ,
Comprising one or more (eg two or more) genetic mutations selected from the group consisting of:

According to a particular embodiment, the bacterium of the invention comprises one or more nucleotide substitutions in thrA that result in the amino acid substitution Y356C in the encoded polypeptide; as well as:
One or more nucleotide substitutions in the Irp gene that result in the amino acid substitution D143G in the encoded polypeptide,
one or more nucleotide substitutions in the rho gene that result in the amino acid substitution R87L in the encoded polypeptide,
One or more nucleotide substitutions in the eno gene that result in amino acid substitution V164L in the encoded polypeptide, and one or more nucleotide substitutions in the argP gene that result in amino acid substitution V164L in the encoded polypeptide. Comprising one or more (eg two or more) genetic mutations selected from the group consisting of:

According to a particular embodiment, the bacterium of the invention comprises in the thrA gene one or more nucleotide substitutions which result in the amino acid substitution S357R in the encoded polypeptide; and:
One or more nucleotide substitutions in the Irp gene that result in the amino acid substitution D143G in the encoded polypeptide,
one or more nucleotide substitutions in the rho gene that result in the amino acid substitution R87L in the encoded polypeptide,
One or more nucleotide substitutions in the eno gene that result in amino acid substitution V164L in the encoded polypeptide, and one or more nucleotide substitutions in the argP gene that result in amino acid substitution V164L in the encoded polypeptide. ,
Comprising one or more (eg two or more) genetic mutations selected from the group consisting of:

According to a particular embodiment, the bacterium of the invention comprises in the thrA gene one or more nucleotide substitutions which result in the amino acid substitution S359R in the encoded polypeptide; and:
One or more nucleotide substitutions in the Irp gene that result in the amino acid substitution D143G in the encoded polypeptide,
one or more nucleotide substitutions in the rho gene that result in the amino acid substitution R87L in the encoded polypeptide,
One or more nucleotide substitutions in the eno gene that result in amino acid substitution V164L in the encoded polypeptide, and one or more nucleotide substitutions in the argP gene that result in amino acid substitution V164L in the encoded polypeptide. ,
Comprising one or more (eg two or more) genetic mutations selected from the group consisting of:

According to a particular embodiment, the bacterium of the invention has in its genome a deletion of the first 5 base pairs of the rhtA gene, a complete deletion of the genes ompX and ybiP, a deletion of the 239 base pair sRNA rybA and 77. It contains a deletion of the base pair gene mntS. According to a particular embodiment, the bacterium comprises in its genome a deletion of about 2854 base pairs from the position corresponding to position 850092 in the genomic sequence NC_000913. This deletion results in a deletion of the first 5 base pairs of the gene rhtA gene, a complete deletion of the genes ompX and ybiP, a deletion of the 239 base pair sRNA rybA and a deletion of the 77 base pair gene mntS. Such deletions can be achieved by the use of Lambda Red or the cam-sacB-system.

According to a particular embodiment, the bacterium of the invention has an insertion of the insertion sequence element IS1 (for example having a length of about 768 base pairs) in its genome in the intergenic region between the genes trxA and rho. Including. According to a particular embodiment, the bacterium has the insertion sequence element IS1 (for example having a length of about 768 base pairs) in the lagging strand at a position in its genome corresponding to position 3966174 in the genomic sequence NC_000913. Having an insert. In a specific embodiment, the bacterium further comprises an overlap about 9 bases upstream and downstream of the insertion sequence.

In a particular embodiment, the bacterium of the invention comprises a single base pair insertion in its genome in the intergenic region between the genes gcvA and ygdl. In a particular embodiment, the bacterium comprises a single base pair insertion in its genome at a position corresponding to position 2942629 in NC_000913 in the genomic sequence.

According to a particular embodiment, the bacterium of the invention has an insertion of the insertion sequence element IS4 (for example having a length of about 1342 base pairs) in its genome in the intergenic region between the genes gcvA and ygdl. Including. According to a particular embodiment, the bacterium comprises an insertion of the insertion sequence element IS4 (for example having a length of about 1342 base pairs) in its genome at a position corresponding to position 2942878 in NC_000913 in the genomic sequence. .. In a specific embodiment, the bacterium further comprises an overlap of about 13 base pairs upstream and downstream of the insertion sequence.

According to a particular embodiment, the bacterium of the invention comprises a single base pair insertion in its genome in the intergenic region between the genes dapA and gcvR. According to a particular embodiment, the bacterium comprises a single base pair insertion in its genome at a position corresponding to position 2599854 in the genomic sequence NC_000913.

According to a particular embodiment, the bacterium of the invention comprises an insertion of the insertion sequence element IS1 (for example having a length of about 768 base pairs) in its genome leading to the truncation of the gene frc. According to a particular embodiment, the bacterium has the insertion sequence element IS1 (for example having a length of about 768 base pairs) in the lagging strand at a position in its genome corresponding to position 2492323 in the genomic sequence NC_000913. Including insert. In a specific embodiment, the bacterium further comprises an overlap of 9 base pairs upstream and downstream of the insertion sequence.

According to another particular embodiment, the bacterium of the present invention is further modified to attenuate the expression of the frc gene (eg by inactivating the gene). Attenuation of gene expression and more specifically inactivation can be achieved as previously described herein. For example, Lambda Red mediated gene exchange may be used to inactivate gene expression.

According to a particular embodiment, the bacterium of the invention comprises an insertion of the insertion sequence element IS5 (for example having a length of about 1195 base pairs) in its genome leading to the deletion of the majority of the gene aroP. According to a particular embodiment, the bacterium comprises an insertion of the insertion sequence element IS5 (for example having a length of about 1195 base pairs) in its genome at a position corresponding to position 121518 in the genomic sequence NC_000913. According to a specific embodiment, the bacterium further comprises an overlap of 4 base pairs upstream and downstream of the insertion sequence.

According to another particular embodiment, the bacterium of the invention is further modified to attenuate the expression of the aroP gene (eg by inactivating the gene). Attenuation of gene expression and more specifically inactivation can be achieved as previously described herein. For example, Lambda Red mediated gene exchange may be used to inactivate gene expression.

According to a particular embodiment, the bacterium of the invention comprises an insertion of the insertion sequence element IS1 (for example having a length of about 768 base pairs) in its genome in the intergenic region between the genes mdtJ and tqsA. ..

According to a particular embodiment, the bacterium inserts in its genome an insertion sequence element IS1 (for example having a length of about 768 base pairs) in the lagging strand at a position corresponding to position 1673670 in the genomic sequence NC_000913. including. In a specific embodiment, the bacterium further comprises an overlap of 9 base pairs upstream and downstream of the insertion sequence.

According to a particular embodiment, the bacterium of the invention comprises in its genome a nucleotide substitution such as a C→T substitution in the intergenic region between the genes trxB and Irp. According to a particular embodiment, the bacterium comprises in its genome a nucleotide substitution, such as a C→T substitution, at a position corresponding to position 923321 in the genomic sequence NC — 000913. Such mutations are 271 base pairs upstream of trxB and 274 base pairs upstream of Irp.

According to a particular embodiment, the bacterium of the invention comprises in its genome a nucleotide substitution, such as a T→C substitution, in the intergenic region between the genes yftB and fklB. According to a particular embodiment, the bacterium comprises in its genome a nucleotide substitution, such as a T→C substitution, at a position in the genomic sequence corresponding to position 4428871 in NC_000913. Such mutations are 154 base pairs upstream of yftB and 64 base pairs upstream of fklB.

As further demonstration herein (eg, by gene inactivation), attenuation of expression of a gene encoding a polypeptide having glucose 6-phosphate-1-dehydrogenase (G6PDH) activity in a reverse engineered strain. (Eg, due to gene inactivation) resulted in significantly increased production and yield of L-serine from glucose, as shown in Table S9 (Example 8).

Therefore, according to a particular embodiment, the bacterium of the present invention has been modified to attenuate the expression of the gene encoding a polypeptide having glucose 6-phosphate-1-dehydrogenase (G6PDH) activity. More specifically, the present invention provides bacteria that have been modified to attenuate expression of the gene zwf. For example, additional information regarding E. coli zwf is available at EcoCyc (www.biocyc.org) under accession number EG11221. A representative nucleotide sequence for zwf is set forth in SEQ ID NO:30.

Gene expression may be attenuated by gene inactivation. Therefore, the bacterium according to the present invention may be one in which a gene encoding a polypeptide having glucose 6-phosphate-1-dehydrogenase (G6PDH) activity is denatured and inactivated (for example, by inactivating the gene, ).

Attenuation of gene expression and more specifically inactivation can be achieved as previously described herein. For example, Lambda Red mediated gene exchange may be used to inactivate gene expression.

According to a particular embodiment, the bacterium of the invention expresses a polypeptide encoded by the brnQ gene, wherein said polypeptide terminates after position 308 or at any position upstream thereof. According to another particular embodiment, the bacterium of the invention is further modified to attenuate the expression of the brnQ gene (eg by inactivating the gene). Attenuation of gene expression and more specifically inactivation can be achieved as previously described herein. For example, Lambda Red mediated gene exchange may be used to inactivate gene expression.

According to a particular embodiment, the bacterium of the invention is further modified to attenuate the expression of a gene encoding a polypeptide having glucose 6-phosphate-1-dehydrogenase (G6PDH) activity; encoded by the thrA gene. Expresses a polypeptide, wherein serine is replaced by arginine at position 357 in said polypeptide; expresses the polypeptide encoded by the rho gene, wherein R at position 87 in said polypeptide Has been replaced by L; and expresses the polypeptide encoded by the brnQ gene, wherein said polypeptide terminates after position 308 or at any position upstream thereof. Alternatively, the bacterium may be denatured to attenuate expression of the brnQ gene (eg, by gene inactivation). Suitable methods of attenuating gene expression have been described above.

For example, additional information regarding E. coli brnQ is available at EcoCyc (www.biocyc.org) under accession number EG12168. A representative amino acid sequence for brnQ is set forth in SEQ ID NO:31.

In a specific embodiment, the bacterium is further modified to attenuate expression of the gene zwf (eg, by inactivating the gene); a polypeptide having the amino acid sequence set forth in SEQ ID NO:11 or SEQ ID NO:11. 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97% of the amino acid sequence of claim 1, wherein serine is replaced by arginine at position 357 of said amino acid sequence. %, at least about 98%, or at least about 99% express a polypeptide having an amino acid sequence having sequence homology;
At least about 90%, at least about 90%, in the polypeptide having the amino acid sequence set forth in SEQ ID NO: 13 or in the amino acid sequence set forth in SEQ ID NO: 13 (where R is replaced by L at position 87 in said amino acid sequence). Expresses a polypeptide having an amino acid sequence having 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence homology; and SEQ ID NO:32. A polypeptide having the amino acid sequence of or at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about at least about 90% of the amino acid sequence set forth in SEQ ID NO:32. Express a polypeptide having an amino acid sequence with 99% sequence homology.

In a specific embodiment, the bacterium is further denatured to attenuate expression of the genes zwf and brnQ (eg, by inactivating the gene); and the amino acid sequence set forth in SEQ ID NO: 11 (wherein said amino acid is Serine at position 357 in the sequence) is substituted with arginine) or at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about the amino acid sequence set forth in SEQ ID NO:11. Expresses a polypeptide having an amino acid sequence having sequence homology of about 97%, at least about 98%, or at least about 99%; a polypeptide having the amino acid sequence set forth in SEQ ID NO:13 or the amino acid set forth in SEQ ID NO:13 At least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98% to the sequence (R is replaced by L at position 87 in said amino acid sequence), Alternatively, one expresses a polypeptide having an amino acid sequence that has at least about 99% sequence homology.

As detailed above, the bacterium of the present invention may be modified to overexpress a particular polypeptide as detailed herein. This means introducing into the bacterium an exogenous nucleic acid molecule, eg a DNA molecule, containing a nucleotide sequence encoding said polypeptide. Techniques for introducing exogenous nucleic acid molecules, such as DNA molecules, into bacterial cells are well known to those of skill in the art and include, among others, transformation (eg, heat shock or natural transformation).

To promote overexpression of the polypeptide in bacteria, the exogenous nucleic acid molecule may include suitable regulatory elements, such as a promoter, which functions in bacterial cells and causes the production of mRNA. , And this is operably linked to the nucleotide sequence encoding the polypeptide.

Promoters useful in accordance with the present invention are any known promoters that function in a given host cell which causes the production of mRNA molecules. Many such promoters are known to those of skill in the art. Such promoters include promoters normally associated with other genes and/or promoters isolated from bacteria. The use of promoters for protein expression is generally known to those skilled in molecular biology (eg, Sambrook et al.; Molecular cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor N. 1982. See). The promoter used may be inducible, eg a temperature inducible promoter (eg pL or pR phage λ promoter, each of which may be controlled by the temperature sensitive λ repressor c1857). ..

The term "inducible" when used in connection with a promoter refers to a nucleotide sequence to which the promoter is operably linked when a stimulus is present, eg in the presence of a change in temperature or the presence of a chemical ("chemical inducer"). Means to guide only the transcription of. As used herein, "chemical induction" according to the present invention refers to the physical application of exogenous or endogenous substances (including macromolecules such as proteins or nucleic acids) to host cells. This has the effect of increasing the transcription rate of the target promoter present in the host cell. Alternatively, the promoter used may be constitutive. The term "constitutive" as used in connection with a promoter means capable of directing the transcription of an operably linked nucleotide sequence in the absence of stimulus (eg, heat shock, chemicals, etc.).

Temperature-inducible systems work, for example, by the use of promoters that are repressed by heat-sensitive repressors. These repressors are active at lower temperatures, eg 30°C. On the other hand, it is inactive because it cannot fold correctly at 37°C. Thus, such circuits can be used to directly control the gene of interest (St-Pierre et al., 2013), or by genomic insertion of the gene with a repressor. Examples of such temperature-inducible expression systems are based on the PL and/or pRλ phage promoters controlled by the heat sensitive C1857 repressor. Similar to the DE system introduced into the genome, expression of the T7 RNA polymerase gene may be controlled by a temperature-regulated promoter system (Mertens et al., 1995), while expression of the gene of interest is controlled by the T7 promoter. Can be controlled using.

Non-limiting examples of promoters that function in bacteria such as E. coli include constitutive and inducible promoters such as the T7 promoter, β-lactamase and lactose promoter systems; alkaline phosphatase (phoA) promoter, tryptophan (trp) promoter system, Included are tetracycline promoters, lambda-phage promoters, ribosomal protein promoters and hybrid promoters such as the tac promoter. Other bacteria and synthetic proteins are also suitable.

In addition to the promoter, the exogenous nucleic acid molecule may further comprise at least one regulatory element selected from the 5'untranslated region (5'UTR) and the 3'untranslated region (3'UTR). Many such 5'UTRs and 3'UTRs derived from prokaryotes and eukaryotes are well known to those of skill in the art. Such regulatory elements include 5'UTRs and 3'UTRs normally associated with other genes and/or 5'UTRs and 3'UTRs isolated from any bacterium.

Usually, the 5'UTR contains a ribosome binding site (RBS), also known as the Shine-Dalgarno sequence, 3-10 base pairs upstream from the start codon.

The exogenous nucleic acid molecule may be a vector or part of a vector, eg an expression vector. Usually such vectors remain extrachromosomal within the bacterial cell. This means that the vector is found outside the bacterial nuclear or nucleoid region.

The present invention also contemplates the stable insertion of exogenous nucleic acid molecules into the bacterial genome. Means for stable insertion into the host cell genome, such as homologous recombination, are well known to those of skill in the art.

The bacterium according to the present invention can be produced from any suitable bacterium, for example a gram positive or gram negative bacterium.

Examples of bacteria that can be used to induce the bacterium of the present invention include genera of the family Enterobacteriaceae, such as Escherichia coli, Arsenophonus, Biostraticola, Brenneria, Buchnera, Budvicia, Buttiauxella, Cedecea, Citrobacter, Cosenzaea, Cronobacter, Dikeinweerweedeacterba, Edwernier, Edwardera, Edwardera, Edwertera Ewingella), Gibbsiella, Hafnia, Klebsiella, Leclercia, Leminorella molla, Londa morrella, Lonsdalea, Lonsdalea, Lonsdalea, Lonsdalea, Mongrovicata. Obesumbacterium, Pantoea, Pectobacteria, Phaseolibacter, Photorhabula, Plesiomonraus, Plesiomonaus, Plesiomonaus, Plesiomonaus Raoultella), Saccharobacter, Salmonella, Samsonia, Serratia, Simwellia, Shimwellia, Sodularis, Sodalis, Tatumera, Tatumella, Tatumella, Tatumella, Tatumella, Tatumella, Tatumella, Tatumella, Tatumella, Tatumella, Tatumella, Tatumella, Tatumella, Tatumella, Tatumella, Tatumella, Tatumella, Tatumella, Tatumella, Tatumella, and Tatumella. , Wigglesworthia, Yersinia, and Yokenella, belonging to a genus selected from the group consisting of:

According to another particular embodiment, the bacterium is Escherichia coli, Bacillus, Lactococcus, Lactobacillus, Clostridium, Corynebacterium, Geobacillus. (Streptococcus), Pseudomonas (Pseudomonas), Streptomyces (Streptomyces), Shigella (Shigella), Acinetobacter (Acinetobacter), Citrobacter (Elberactera) (Citrobacter elabenella), Salmonella (Kelbactera), Salmonella (Salmonella), Salmonella (Salmonella), Salmonella (Salmonella, Clemonella) Erwinia, Kluyvera, Serratia, Cedecea, Morganella, Hafnia, Edwainella (Edwardersiella), Providencia (Proteus and Protedeniusia), and Providencia (Providenusia). Belong to a genus selected from the group.

In a specific embodiment, the bacterium belongs to the genus Escherichia coli. In a specific embodiment, the bacterium is E. coli. A non-limiting example of a bacterium belonging to the genus Escherichia coli that can be used to induce the bacterium of the present invention is E. coli K-12 (particularly substrain MG1655 or W3110), BL21, W, or Brooks. In a more specific embodiment, the bacterium is E. coli K-12.

In a specific embodiment, the bacterium belongs to the genus Corynebacterium. A non-limiting example of a bacterium of the genus Corynebacterium is Corynebacterium glutamicum. In a more specific embodiment, the bacterium is Corynebacterium glutamicum.

According to another particular embodiment, the bacterium belongs to the genus Bacillus. Non-limiting examples of Bacillus bacteria are Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus moyabensis. In a more specific embodiment, the bacterium is Bacillus subtilis. In another more specific embodiment, the bacterium is Bacillus licheniformis.

In a specific embodiment, the bacterium belongs to the genus Lactococcus. A non-limiting example of a Lactococcus bacterium is Lactococcus lactis. In a more specific embodiment, the bacterium is Lactococcus lactis.

According to another particular embodiment, the bacterium belongs to Streptomyces. Non-limiting examples of Streptomyces bacteria are Streptomyces lividans, Streptomyces sericara, or Streptomyces griseus. In a more specific embodiment, the bacterium is Streptomyces lividans. In another more specific embodiment, it is Streptomyces coelicolor. In another more specific embodiment, the bacterium is Streptomyces griseus.

According to another particular embodiment, the bacterium belongs to the genus Pseudomonas. A non-limiting example of a Pseudomonas bacterium is Pseudomonas putida. In a more specific embodiment, the bacterium is Pseudomonas putida.

Method of the present invention The present invention also provides a method for producing L-serine or an L-serine derivative using the bacterium according to the present invention. In particular, the invention provides a method for producing L-serine or an L-serine derivative, said method comprising culturing a bacterium in a medium as detailed herein.

According to a particular embodiment, the present invention provides a method for producing L-serine. In particular, the present invention provides a method for producing L-serine, which method comprises culturing a bacterium as detailed herein in a medium. The method may further include recovering L-serine from the medium.

According to a particular embodiment, the present invention provides a method for producing an L-serine derivative. Especially. The present invention provides a method for producing an L-serine derivative, the method comprising culturing a bacterium as detailed herein in a medium. The L-serine derivative may be selected from the group consisting of L-cysteine, L-methionine, L-glycine, O-acetylserine, L-tryptophan, thiamine, ethanolamine and ethylene glycol. The method may further include recovering the L-serine derivative from the medium.

According to a particular embodiment, the present invention provides a method for producing L-cysteine. In particular, the invention provides a method for producing L-cysteine, said method comprising culturing a bacterium as detailed herein in a medium. The method may further include recovering L-cysteine from the medium.

According to a particular embodiment, the present invention provides a method for producing L-methionine. In particular, the present invention provides a method for producing L-methionine, said method comprising culturing a bacterium as detailed herein in a medium. The method may further include recovering L-methionine from the medium.

According to a particular embodiment, the present invention provides a method for producing L-glycine. In particular, the invention provides a method for producing L-glycine, said method comprising culturing a bacterium as detailed herein in a medium. The method may further include recovering L-glycine from the medium.

According to a particular embodiment, the present invention provides a method for producing O-acetylserine. In particular, the present invention provides a method for producing O-acetylserine, which method comprises culturing a bacterium as detailed herein in a medium. The method may further include recovering O-acetylserine from the medium.

According to a particular embodiment, the present invention provides a method for producing L-tryptophan. In particular, the present invention provides a method for producing L-tryptophan, said method comprising culturing a bacterium as detailed herein in a medium. The method may further include recovering L-tryptophan from the medium.

According to a particular embodiment, the present invention provides a method for producing thiamine. In particular, the invention provides a method for producing thiamine, said method comprising culturing a bacterium as detailed herein in a medium. The method may further include recovering thiamine from the medium.

According to a particular embodiment, the present invention provides a method for producing ethanolamine. In particular, the invention provides a method for producing ethanolamine, said method comprising culturing a bacterium as detailed herein in a medium. The method may further include recovering ethanolamine from the medium.

According to a particular embodiment, the present invention provides a method for producing ethylene glycol. In particular, the present invention provides a method for producing ethylene glycol, said method comprising culturing a bacterium as detailed herein in a medium. The method may further include recovering ethylene glycol from the medium.

The medium used may be any conventional medium suitable for culturing the bacterial cells in question and may be constructed according to the principles of the prior art. The medium will normally contain all the nutrients necessary for the growth and survival of the individual bacteria, such as carbon and nitrogen sources and other inorganic salts. Suitable media, such as minimal or complex media, are available from commercial suppliers or may be prepared according to published recipes, eg, the strain catalog of the American Type Culture Collection (ATCC). Non-limiting standard media known to those of skill in the art include Luria Bertani (LB) media, Sabouraud dextrose (SD) media, MS media, yeast peptone dextrose, BMMY, GMMY or yeast malt extract (YM) media, which are Are all commercially available. Non-limiting examples of suitable media for culturing bacterial cells such as E. coli cells include minimal and rich media such as Luria broth (LB), M9 media, M17 media, SA media, MOPS media, Terrific media, YT and others are included.

To further increase the yield of L-serine or L-serine derivative, the medium may be supplemented with L-threonine. The medium generally contains L-threonine at a concentration of about 0.05 to about 10 g/L, such as a concentration of about 0.05 to about 5 g/l, about 0.05 to about 2.5 g/L. It may be included at a concentration of about 0.05 to about 1 g/L or at a concentration of about 0.05 to about 0.5 g/L. In a particular embodiment, the medium contains L-threonine at a concentration of about 0.05 to about 5 g/L. According to another particular embodiment, the medium contains L-threonine, for example at a concentration of about 0.05 to about 2.5 g/L. In another particular embodiment, the medium contains L-threonine at a concentration of about 0.05 to about 1 g/L. In another particular embodiment, the medium contains L-threonine, for example at a concentration of about 0.05 to about 0.5 g/L. In another particular embodiment, the medium contains L-threonine at a concentration of about 0.1 to about 1 g/L. According to another particular embodiment, the medium contains L-threonine, for example at a concentration of about 0.2 to about 1 g/L.

The carbon source may be any suitable carbon substrate known in the art and in particular any carbon substrate commonly used in bacterial culture and/or fermentation. Non-limiting examples of suitable fermentable carbon substrates are pentoses (arabinose or xylose), hexoses (eg glucose), acetates, glycerol, vegetable oils, yeast extracts, peptones, casamino acids or mixtures thereof. .. A carbon source of particular interest is a hexose sugar such as glucose.

As the nitrogen source, various ammonium salts such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, neutral nitrogen sources such as peptone, soy hydrolyzate, and digestive fermenting microorganisms can be used. As minerals, potassium phosphate, magnesium sulfate, sodium chloride, iron sulfate, manganese sulfate, calcium chloride and the like can be used.

The culturing can advantageously be carried out under aeration conditions, for example at a temperature of about 20 to about 40° C., for example at about 30 to 38° C., preferably at about 37° C. by shaking culture and with aeration. It can be performed by stirring culture. The pH of the culture is usually about 5 to about 9, for example about 6.5 to 7.5. The pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases and buffers. Usually, a culture for 1 to 5 days results in the accumulation of L-serine in the medium.

After culturing, solids such as cells can be removed from the medium by centrifugation or membrane filtration. The L-serine or L-serine derivative can be recovered from the medium by conventional methods for isolating and purifying chemical compounds. Well-known purification methods include, but are not limited to, centrifugation or filtration, precipitation methods, ion exchange, chromatographic methods such as ion exchange chromatography or gel filtration chromatography and crystallization methods. Be done.

Accordingly, the present invention provides L-serine or L-serine derivatives obtainable by the methods detailed herein.

OTHER SPECIFIC DEFINITIONS As used herein, the term "a bacterium capable of producing L-serine" means a bacterium capable of producing L-serine in a medium and resulting in accumulation. An amount of accumulation of at least 0.4 g/L when the bacterium was cultured at 37° C. for 40 hours with sufficient aeration in minimal M9 medium supplemented with 2 g/L glucose, 2 mM glycine and 1 mM threonine. Means that can occur.

As used herein, the phrase "a bacterium modified to attenuate expression of a gene encoding a polypeptide having serine deaminase activity" means that the modified bacterium has a polypeptide having serine deaminase activity. This means that the bacterium has been modified to contain a small amount of peptide. More specifically, this phrase means that the bacterium is unable to synthesize a polypeptide having serine deaminase activity.

As used herein, the phrase "a bacterium modified to attenuate expression of a gene encoding a polypeptide having serine hydroxymethyl transferase activity" means that the modified bacterium is a serine hydroxymethyl transferase. It means that the bacterium has been modified to contain a small amount of active polypeptide. More specifically, this phrase means that the bacterium is unable to synthesize a polypeptide having serine hydroxymethyl transferase activity.

As used herein, the phrase "a bacterium modified to attenuate expression of a gene encoding a polypeptide having glucose 6-phosphate-1-dehydrogenase (G6PDH) activity" is modified It means that the bacterium has been modified so that it contains a polypeptide with a small amount of glucose 6-phosphate-1-dehydrogenase (G6PDH). More specifically, this phrase means that the bacterium is unable to synthesize a polypeptide having glucose 6-phosphate-1-dehydrogenase (G6PDH) activity.

As used herein, the phrase "yeast modified to attenuate the expression of a gene encoding a polypeptide having serine deaminase activity" refers to a modified yeast having a polypeptide having serine deaminase activity. This means that the yeast was modified to contain a small amount of peptide. More specifically, this phrase means that yeast is unable to synthesize a polypeptide having serine deaminase activity.

As used herein, the phrase "yeast modified to attenuate expression of a gene encoding a polypeptide having serine hydroxymethyl transferase activity" means that the modified yeast is a serine hydroxymethyl transferase. It means that the yeast was modified to contain a small amount of active polypeptide. More specifically, this phrase means that yeast is unable to synthesize a polypeptide having serine hydroxymethyl transferase activity.

The phrase "gene inactivation" can mean that the denatured gene encodes a completely nonfunctional protein. In addition, deletion of a part or the whole of the gene sequence, shift of the reading frame of the gene, introduction of missense/nonsense mutations, or flanking regions of the gene (sequences that control gene expression, such as promoters, enhancers, (Including an attenuator, a ribosome binding site, etc.) can also prevent a denatured DNA region from naturally expressing a gene. Advantageously, the gene of interest is inactivated by partial or total deletion of the entire gene sequence, eg gene exchange.

The presence or absence of a gene on a bacterial chromosome can be detected by well known methods including PCR, Southern blotting and the like. In addition, gene expression levels can also be assessed by measuring the amount of mRNA transcribed from the gene, using a variety of well-known methods including Northern blotting, quantitative RT-PCR and the like. The amount of protein encoded by the gene can be measured by well known methods including SDS-PAGE followed by immunoblot assay (Western blotting analysis) and the like.

The terms "polypeptide" and "protein" are used interchangeably herein, regardless of length or post-translational modification (eg, glycosylation, phosphorylation, lipidation, myristylation, ubiquitination, etc.). By a polymer of at least two amino acids covalently linked by an amide bond. Included within this definition are D-amino acids and L-amino acids and mixtures of D-amino acids and L-amino acids.

"Nucleic acid" or "polynucleotide" are used interchangeably herein and refer to at least two nucleic acid monomer units or bases covalently linked by a phosphodiester bond, regardless of length or base modifications (eg, Adenine, cytosine, guanine, thymine).

For example, when the term "recombinant" or "non-naturally occurring" is used in reference to a host cell, nucleic acid or polypeptide, material modified in a manner that does not occur in nature, or material equivalent to nature or It refers to the natural form of the material, or equivalent but produced or derived from synthetic material and/or modified by manipulation using recombinant techniques. Non-limiting examples include, inter alia, recombinant host cells that express genes not found in the native (non-recombinant) form of the cell, or expression of native genes that would otherwise be expressed at different levels.

As used herein, "heterologous" means that a polypeptide is not normally found in or produced by (or is expressed by) the host organism, but is derived from a different species. means.

"Substitution" or "substituted" refers to modification of a polypeptide by exchanging one amino acid residue for another, eg, a serine residue and a glycine or alanine residue in a polypeptide sequence. The group exchange is an amino acid substitution. As used in the context of polynucleotides, “substitution” or “substituted” refers to the modification of a polynucleotide by exchanging one nucleotide for another, eg, in a polynucleotide sequence The exchange of cytosine and thymine at is a nucleotide substitution.

As used in the context of polypeptides, "conservative substitution" means the substitution of an amino acid residue with a different residue having a similar side chain. Thus, it is usually involved in substituting amino acids in a polypeptide with amino acids in the same polypeptide or with similar classes of amino acids. By way of example and not limitation, amino acids having an aliphatic side chain can be replaced with other aliphatic amino acids (eg alanine, valine, leucine and isoleucine); amino acids having a hydroxyl side chain have a hydroxyl side chain. Can be substituted with other amino acids (eg serine and threonine); Amino acids with aromatic side chains can be substituted with other amino acids with aromatic side chains (eg phenylalanine, tyrosine, tryptophan and histidine); basic side Amino acids with chains can be replaced with other amino acids with basic side chains (eg, lysine and arginine); amino acids with acidic side chains can be replaced with other amino acids with acidic side chains (eg, aspartic acid). Or glutamic acid); and hydrophobic or hydrophilic amino acids are replaced with other hydrophobic or hydrophilic amino acids, respectively.

"Non-conservative substitution" when used in the context of a polypeptide means a substitution of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitutions may be used between defined groups, rather than within defined groups, and (a) the structure of the peptide backbone at a location surrounding the substitution (eg serine to glycine), (b ) Affects charge or hydrophobicity, or (c) side chain size. By way of example and not limitation, exemplary non-conservative substitutions are acidic amino acids substituted with basic or aliphatic amino acids; aromatic amino acids substituted with small amino acids; and hydrophilic amino acids substituted with hydrophobic amino acids. You can

When "deleted" or "deleted" is used in the context of a polypeptide, it refers to modification of the polypeptide by removal of one or more amino acids in the reference polypeptide. Deletions are removals of one or more amino acids, two or more amino acids, five or more, while retaining enzyme activity and/or retaining the improved properties of the engineered enzyme. Removal of many amino acids, 10 or more amino acids, 15 or more amino acids, 20 or more amino acids, removal of up to 10% of the total number of amino acids forming a polypeptide, Including up to 20% of the total number of amino acids. Deletions can dominate the inner and/or terminal portions of the polypeptide, and in various embodiments, the deletion can include contiguous segments or can be discontinuous.

When "inserted" or "inserted" is used in reference to a polypeptide, it refers to modification of the polypeptide by the addition of one or more amino acids to a reference polypeptide. The insertion may be one or more amino acids, two or more amino acids, five or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20. The addition of one or more amino acids can be included. The insertion can be in the inner portion of the polypeptide or at the carboxy or amino terminus. Insertions may be contiguous segments of amino acids or separated by one or more amino acids in the reference polypeptide.

"Expression" includes any process involved in the production of a polypeptide (eg, an encoded enzyme), including, but not limited to, transcription, posttranscriptional denaturation, translation, posttranslational denaturation and secretion. included.

As used herein, "vector" means a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been attached. One type of vector is a "plasmid", which is a circular double stranded nucleic acid loop to which additional nucleic acid segments can be ligated. Certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors". Other particular vectors can facilitate the insertion of exogenous nucleic acid molecules into the bacterial genome. Such vectors are referred to herein as "transformation vectors." In general, the vectors utilized in recombinant nucleic acid technology are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably. That is because plasmids are the most commonly used form of vector. Large numbers of suitable vectors are known to those of skill in the art, and are commercially available.

As used herein, “promoter” means a sequence of DNA, usually upstream (5′) of the coding region of a structural gene, which is necessary for initiation of RNA polymerase and transcription. Providing recognition and binding sites for waxes and other factors controls expression of the coding region. The choice of promoter will depend on the nucleic acid sequence of interest. A suitable “promoter” is generally one that is capable of supporting initiation of transcription in the bacterium of the present invention and causing the production of mRNA molecules.

As used herein, “operably linked” means proximally in a relationship that enables the components described to function in their intended manner. A control sequence "operably linked" to a coding sequence is achieved under the condition that the coding sequence matches the control sequence. A promoter sequence "operably links" a gene to control transcription of the gene when it is sufficiently close to the transcription start site of the gene.

"Percentage of sequence homology", "% sequence homology" and "percent homology" are used herein to refer to a comparison between an amino acid sequence and a reference amino acid sequence.

"% sequence homology", as used herein, is calculated from two amino acid sequences as follows: 12 using the Genetic Computing Group's GAP (Global Alignment Program), Sequences are aligned using a default BLOSUM62 matrix (see below), with a gap open penalty of 1 (in the first gap of the gap) and a gap extension penalty of -4 (in each additional gap in the gap). After alignment, the percentage homology was calculated by displaying the number of matches as a percentage of the number of amino acids in the reference amino acid sequence.

The following BLOSUM62 matrix was used:

By "reference sequence" or "reference amino acid sequence" is meant a defined sequence in which other sequences are compared. In the context of the present invention, the reference amino acid sequence may be, for example, the amino acid sequence set forth in SEQ ID NO: 5 or 6.

As used herein, "L-serine derivative" refers to an amino acid that results from reaction at the amino or carboxy group of L-serine or from the exchange of any hydrogen for a heteroatom of L-serine. Such compounds are meant. Non-limiting examples of "L-serine derivatives" include L-cysteine, L-methionine, L-glycine, O-acetylserine, L-tryptophan, thiamine, ethanolamine and ethylene glycol. Further examples of "L-serine derivatives" are described by Chemical Entities of Biological Interests (ChEBI) [https://www.ebi.ac.uk/chebi/init.do], eg ChEBI ID CHEBI 84. Has been done.

Where numerical limits or ranges are stated herein, the endpoints are included. Also, all numerical values and subranges or ranges within numerical limits are expressly included as if explicitly set.

Having generally described the invention, further understanding may be obtained by reference to specific specific embodiments. They are provided herein for illustrative purposes only and are not intended to be limiting unless otherwise stated.

EXAMPLES The present inventors have shown for the first time that a bacterium such as Escherichia coli deficient in all four serine degrading genes (sdaA, sdaB, tdcG and glyA) could be constructed (Example 1). The strain showed higher serine production yields than single and triple deaminase knockouts when the serine pathway was upregulated (Example 2). However, the resulting strain had very low resistance to serine, which was also reported in the E. coli triple deletion strain in which sdaA, sdaB and tdcG were deleted (Zhang and Newman, 2008). The inventors further demonstrated that overexpression of the novel exporter (Example 3), evolution of the strain by random mutation (Example 4) and adaptive evolution (Example 5) can reduce the toxicity of the product. did. In addition, the strain was reverse engineered to identify the causative mutation (Example 6). During fed-batch fermentation, the resistant strains showed improved serine production when compared to the parental quadruple deletion strain (12.6 g/L, mass yield from glucose 36.7%) ( Example 7). This is the highest serine mass yield from glucose ever reported in a production organism.

Furthermore, the inventors demonstrated that in the presence of other causative mutations, inhibition of the pentose phosphate pathway by deletion of zwf led to a further increase in serine production yield (Example 8).

Example 1-Deletion of Key Degradation Pathways Serine has two important degradation pathways in E. coli: conversion of serine to pyruvate, which is encoded by three deaminase, sdaA, sdaB and tdcG. .. And the conversion of serine to glycine, which is encoded by glyA. The deletion of glyA renders E. coli auxotrophic for glycine. Deletion of sdaA, sdaB and tdcG was quantitatively performed by using the Lambda Red mediated gene exchange method (Datsenko and Wanner, 2000). The protocol applied for the deletion of these genes is similar to the protocol described by Sawitzke et al. (2013). The primers used to amplify the kanamycin cassette are shown in Table S1. The PCR reaction contained 250 nM of KF and KR primers of a given gene, 250 μM of dNTPmix, 4 units of Phusion polymerase (Thermoscientific, Waltham, MA, USA), 40 μl of HF buffer and 10 ng of pkD4 plasmid. The following two-step PCR protocol was used for PCR amplification: initial denaturation step, 98° C. for 40 seconds, followed by 5 cycles of 98° C. for 10 seconds denaturation, 55° C. for 30 seconds annealing, 72° C. for 90 seconds. Extending for seconds, followed by 20 cycles of this, the annealing temperature was increased from 55°C to 65°C. The PCR products were column purified (Macherey Nagel, Durn, Germany) and the concentrations were measured using the Nanodrop instrument (Thermoscientific, Waltham, MA, USA) and digested overnight with Dpnl. E. coli MG1655 was used as a parent strain to generate continuous knockouts. The parental strain was transformed with pkD46 and grown in 2YT-amp medium at 30° C. and 250 rpm. Expression of exo, β and γ proteins was induced by the addition of 20 mM arabinose in mid-log phase (OD 0.4-0.5) and cells were harvested after an additional 1 h of incubation. The culture was then transferred to 50 ml ice-cold Falcon tubes and centrifuged for 5 minutes at 6500 rpm and 4°C. The supernatant was discarded and the cells were washed twice with ice cold 10% glycerol. These electrocompetent cells were transformed with 200 ng of kanamycin cassette and transformants were plated on LB-kan plates. The kanamycin cassette was removed using the plasmid pcp20, which encodes the flippase gene. Primers that check for loopout are shown in Table 2. The serine hydroxymethyltransferase encoded by glyA was detected using the P1 phage-based protocol (Thomason et al., 2007). A strain from the Keio collection (Baba et al., 2006) populating glyA::kan was grown in 5 ml LB-kan supplemented with 0.2% glucose, 25 mM CaCl ₂ and 1 mM glycine medium. In the early log phase (OD 0.1), cultures were transformed with P1 phage lysate. Cultures were incubated for 3 hours at 37° C. and 250 rpm for cell lysis and cells were lysed at 250 rpm. The lysate was sterile filtered by using a 0.45 μM filter. 1 ml of an overnight culture of an E. coli strain deficient in sdaA, sdaB and tdcG was resuspended in 200 μL of P1 salt solution and incubated with 100 μL of the lysate for 1 hour. The cells were then grown overnight in 2 mL LB medium supplemented with 2 mM glycine and 200 mM sodium citrate. Cells were plated on LB-kan plates supplemented with 2 mM glycine and 10 mM sodium citrate. To avoid phage contamination, clones were restreaked on new plates and isolated clones were checked for cassette insertion. Loopout was performed using the pcp20 plasmid. The resulting quadruple deletion strain (FIG. 1) is designated Q1. This example demonstrates that sdaA, sdaB, tdcG and glyA can be deleted in E. coli. This has never been accomplished before and was unpredictable.

Table S1: Primers used for amplification of kanamycin cassette

Table S2: Primers used to check for deletion of a given gene

Example 2-Overexpression of the serine biosynthetic pathway to produce serine Serine was produced in E. coli from three enzymes encoded by serA, serB and serC. All genes were isolated from E. coli MG1655 using primers with the respective gene name (Table S3). A 100 μl PCR mix contained 250 nM of forward and reverse primers, 250 μM of dNTP, 2 units of Phusion polymerase, 1×HF buffer, 1 μl of overnight culture, respectively. The following two-step PCR protocol was used for PCR amplification: initial denaturation step, 98° C. for 40 seconds, followed by 5 cycles of 98° C. for 10 seconds of denaturation, 55° C. for 30 seconds annealing, 72° C. for 90 seconds. Elongation, followed by 20 cycles, with the annealing temperature increased from 55°C to 65°C. After column purification, the gene product and plasmid were digested using Fast digest enzyme (Thermoscientific, Waltham, MA, USA). About 500 ng of PCR product or 1 μg of plasmid was digested with 1 μl of each restriction enzyme in 1×fast digest buffer. The reaction was incubated for 3 hours and then column purified again. SerA was double digested with NcoI and NotI, while serC was digested with NdeI and PacI. First, pCDF-Duet was digested with NcoI and NotI so that cloning of serA yielded the plasmid pCDF-Duet-serA, and this plasmid was later used for cloning of serC, thus pCDF-Duet-serA-serC. Was produced. The gene encoding serB at the NcoI and PacI sites was cloned into the pACYC-Duet vector resulting in pACYC-serB. Typical ligation reactions include 1×T4 ligase buffer, 50 ng plasmid DNA and 100 ng insert and T4 DNA ligase (Thermoscientific, Waltham, MA, USA) 0.3 μl/10 μl.

Feedback inhibition of serA was eliminated by mutating three residues H344, N346 and N364 to alanine by site-directed mutagenesis (Al-Rabiee et al., 1996) (Table S4). The master mix was used as above, with the only change being that the master mix was divided into two equal aliquots. Following this, forward and reverse primer primers were added to each aliquot. A total of 100 ng of pCDF-Duet-serA-serC plasmid was used as template. Two-step PCR program: initial denaturation at 98°C for 40 seconds, denaturation at 98°C for 10 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 4 minutes and 30 seconds, and this cycle repeated 5 times, The two aliquots were then combined and redistributed for an additional 15 cycles. To allow for vector backbone replacement, the NcoI site inside serC was removed by the same approach using the primers listed in Table S4.

FIG. 2 shows a vector map of the plasmid construct.

Table S3: Primers used for amplification and cloning of the serine production pathway

Table S4: Primers used for site-directed mutagenesis of the serine production pathway

DE3 Insertion and Serine Production During Batch Fermentation Serine production was checked in M9 minimal medium. Glucose M9 minimal medium consists of 2 g/L glucose, 0.1 mM CaCl ₂ , 2.0 mM MgSO ₄ , 1× trace element solution, and 1×M9 salt. 4000 x trace element stock solution is 27 g/L FeCl ₃ *6H ₂ O, 2 g/L ZnCl ₂ *4H ₂ O, 2 g/L CoCl ₂ *6H ₂ O, 2 g/L NaMoO ₄ *2H ₂ O, 1 g/L It consisted of L CaCl ₂ *H ₂ O, 1.3 g/L CuCl ₂ *6H ₂ O, 0.5 g/L H ₃ BO ₃ and concentrated HCl dissolved in ddH ₂ O and was sterile filtered. The 10×M9 salt stock solution consisted of 68 g/L anhydrous Na ₂ HPO ₄ , 30 g/L KH ₂ PO ₄ , 5 g/L NaCl and 10 g/L NH ₄ Cl dissolved in ddH ₂ O and was autoclaved.

To use the pET vector as an expression system, the T7 polymerase containing DE3 cassette was inserted into the genome of each deletion mutant using the Lambda DE3 lysogenization kit (Millipore, Damstadt, Germany). Subsequently, strains (MG1655(DE3)) with single (ΔsdaA), triple (ΔsdaA, ΔtdcG, ΔsdaB) and quadruple deletions (ΔsdaA, ΔtdcG, ΔsdaB and ΔglyA) were pCDF-Duet1-serAmut-serC, pACY and pACY. It was transformed with. The resulting glycerol stock was grown overnight in 2YT medium containing 0.1% glucose and supplemented with the required antibiotics (spectinomycin and chloramphenicol). Overnight cultures were plated in triplicate in flasks containing M9 medium supplemented with 0.2% glucose, 1 mM threonine, the required antibiotics (quadruple deletion strain was also supplemented with 2 mM glycine). The flask was incubated at 37° C. and 250 rpm. After the culture reached an optical density of OD 0.55-0.65, serine production was induced by the addition of 40 μM IPTG. O.D. measurements and samplings were then taken at regular time intervals for 60 hours. Samples were filtered using the method described above, except that the column temperature was kept at 30° C., and HPLC was performed on glucose and by-products (Kildegaard et al., 2014). Serine concentration was measured using LCMS. The LC-MS/MS system consists of a CTC autosampler module, a high pressure mixing pump and a column module (Advance, Bruker, Fremont, CA, USA). The injection volume was 1 μl. Chromatography was performed on a ZIC-cHILIC column, 150 mm×2.1 mm, pore size 3 μm (SeQuant, Merck Millipore). In front of the separation column was a 0.5 μ depth filter and guard column, a filter (KrudKatcher Classic, phenomenex) and a guard column ZIC-cHILIC, 20×2.1 mm (SeQuant, Merck). Eluent A: 20 mM ammonium nitrate (pH adjusted to 3.5 with formic acid in milliQ). Eluent B: acetonitrile. The total flow rate of the eluates A and B was 0.4 ml/min. Elution of homogeneous solvent 35% and total run time was 5 minutes. The residence time was 2.8 minutes for serine. MS-MS detection was performed on an EVOQ triple quadrupole instrument (Bruker, California, USA) equipped with an atmospheric pressure ionization (API) interface. The mass spectrometer was operated with electrospray in positive ion mode (ESI+). The spray pressure was adjusted to 4500V. The cone gas flow was 20 liters/h and the cone temperature was adjusted to 350°C. The temperature was 350° C. and the heated probe gas flow was adjusted to 50 l/h. The Nebulizer flow was adjusted to 50 l/h and the exhaust gas was emitted. Argon was used as the collision gas at a pressure of 1.5 mTorr. Detection was performed in the combined reaction detection (MRM) mode. The quantitative transfer was 106→60 and the qualitative transfer was 106→70. The collision energy was optimized to 7 eV for both transfers. A calibration standard for serine was prepared in the medium used for serine production and diluted 50:50 with 0.2% formic acid in acetonitrile. The concentration of the calibration standard was in the range of 0.001 to 0.5 g/L.

As shown in Figure 3, this experiment showed that all four genes involved in serine degradation in E. coli, designated Q1(DE3), produced the highest specific productivity from glucose and the highest yields. Demonstrate what happened.

Example 3: Reduction of Product Toxicity by Overexpression of Transporters Lack of the major serine degradation pathway in E. coli resulted in significantly reduced resistance to serine. To increase resistance, a transporter with potential specificity for serine (ydeD) was overexpressed and tested during growth of high concentrations of serine. Using the primers listed in Table S3 and the protocol listed in Example 1, ydeD was cloned in pCDF-1b by amplification at the NcoI and PacI sites.

Q1(DE3) was transformed with pCDF-1b empty vector and pCDF-ydeD-c-His plasmid (FIG. 4). Transformants were selected on LB-spectinomycin plates. Overnight cultures of these transformants were seeded in M9 medium supplemented with 2 mM glycine, 2 g/L glucose and spectromycin (ratio 1:50). The cultures were incubated at a broad optical density of 0.4-0.5 at 37° C. and 250 rpm, after which 800 μl containing 800 μl of M9 medium with various serine concentrations (12.5, 25 and 50 g/L). Added to 48-well Biolector plates (M2P labs, Baeswierer, Germany). Expression of ydeD was induced with 100 μM IPTG. It was then monitored in a Biolector device (M2P labs, Baeswierer, Germany) at 37° C. and 70% humidity with continuous shaking. The resulting one was set to 20% and the scattering intensity was measured every 5 minutes for the next 4 hours.

This experiment demonstrated that the presence of low concentrations of serine significantly inhibited the growth of E. coli lacking the major serine degradation pathway. Upon overexpression of ydeD, resistance to serine was substantially increased (Fig. 4), suggesting that YdeD may have exported serine from the cells.

Example 4-Random mutations on serine resistant strains Inactivation of the major serine degradation pathway in E. coli resulted in significantly reduced resistance to serine. Strain Q1 (obtained in Example 1) was grown overnight in M9 medium supplemented with 2 mM glycine and 3 g L-serine to increase resistance. 1 ml of medium was spread in a 6-well Petri dish and exposed to UV irradiation for 30 minutes (CBS Scientific, San Diego, CA, USA). The cultures were then added to 5 ml M9 medium supplemented with 2 mM glycine, 2 g/L glucose and 50 g/L serine to multiply resistant mutants. The cultures were incubated at 37° C. and 250 rpm for 3 days and then plated on M9 plates supplemented with 50 g/L serine for selection of resistant clones. Colonies were isolated and growth rate and resistance estimated using the following method:

Cultures were grown overnight in 2xYT medium. They were then seeded in 96-well flat bottom microtiter plates with 150 μL of M9 medium containing 0.2% glucose and varying concentrations of serine (in triplicate). A total of 1.5 μL cells (1:100) were used as inoculum. If necessary, small changes in volume were made to ensure that the cells were equal. Plates were sealed with Microamp clear adhesive film (Applied Biosystems, Warrington, UK) and incubated in a microtiter plate reader (Biotek, Winooski VT, USA). The reader was set at 37° C. with continuous shaking and the OD was monitored at 630 nm every 5-10 minutes for 32 hours.

This experiment demonstrated that random mutations can be used to significantly increase resistance to serine (Figure 5). The obtained strain is hereinafter referred to as Q3.

Example 5: Adaptive evolution for L-serine resistance It was demonstrated that, unlike random mutations, the serine resistance of strains in which the major serine degradation pathway is inactivated can also be increased by adaptive evolution. Seven independent populations of strain Q1 were adaptively evolved in M9 minimal medium supplemented with 2 mM glycine and the concentration of amino acid L-serine was increased at 37° C. for 60 days.

Adaptive laboratory evolution (ALE) experiments are accomplished using an automated system, which allows breeding of populations that evolve over days while monitoring their growth rate. Prior to the start of the experiment, the system was filled with tubes containing 25 ml of the required medium and kept at 37°C in a heat block. Controlled aeration was obtained using a magnetic tumble stirrer placed inside the tube and spinning at 1800 rpm. At the start of the experiment, a single colony grew overnight in one of the tubes placed inside the system, and a 100 μL aliquot was used by the robotics platform to seed seven independent flasks. did. As the bacteria grew, the automated system made multiple OD measurements at 600 nm in each flask. As the bacteria grew, multiple OD measurements were taken at 600 nm. Growth rates were automatically calculated by taking the slope of the least squares linear regression line that fits the log of the OD measurements. Once the target OD reached 0.4, 100 μl of culture was used to seed a new tube. Thus, after reaching the target cell density, which never reached stationary phase, the culture was continuously (2 to 3 times a day) passed through a flask with fresh medium. After reaching the desired growth rate, the population was first incubated with 3 g/L serine and the cultures supplemented with 6 g/L L-serine. L-serine concentration increased to 12 g/L when the population reached a stable phenotype (ie growth rate). This method was repeated using 24, 50, 75 and 100 g/L L-serine. The final population was plated on LB-Agar and two clones of each population were selected. The evolved strains had significantly increased resistance when tested for growth in the presence of high concentrations of serine, using the MTP-based assay described above in Example 5. Results are shown in FIG.

Genomic DNA Extraction, Library Sequencing and Analysis Two clones of each evolved culture as well as a strain evolved by random UV-mutation (Example 5) were genomic sequenced. Genomic DNA was extracted from 1.5 ml of an overnight culture of an E. coli strain using the QIAamp DNA Mini Kit (QIAGEN, Germany). Genomic library was prepared using a TruSeq ^(R) Nano DNA LT sample preparation kit (Illumina InC, San Diego CA, USA).

Briefly, 100 ng of genomic DNA diluted in 52.5 μl TE buffer was duty cycled at 5%, 175 W peak incident power, 200 cycles/burst and 5.5-6° C. for 50 seconds under frequency sweep mode. Was fragmented in Covaris Crim Cap Microtubes in a Covaris E220 Ultrasonicator (Covaris, Brighton, UK) with a residence time of (Illumina's recommendation for an average fragment size of 350 base pairs). The ends of the fragmented DNA were repaired with T4 DNA polymerase, Klenow DNA polymerase and T4 polynucleotide kinase. The Klenow exo minus enzyme was used and then added to the 3'end "A" base of the DNA fragment. After ligation of the adapter to the ends of the DNA fragment, DNA fragments in the range of 300-400 bases were recovered by bead purification. Finally, the adapter denatured DNA fragment was amplified by 3 cycles-PCR. The final concentration of each library was determined by Qubit ^(R) 2.0 Florometer and Qubit DNA Wide Range Assay (Life Technologies, Paisly, UK). Average dsDNA library size was determined on an Agilent 2100 Bioanalyzer using the Agilent DNA 7500 kit. The library was standardized and pooled in 10 mM Tris-Cl, pH 8.0 and 0.05% Tween 20 was added to give a final concentration of 10 nM. Denatured in 0.2 N NaOH and loaded a 10 pM pool of 20 libraries in 600 μl ice-cold HT1 buffer onto a flow cell prepared with MiSeq Reagent kit v2 300 cycles and using a paired-end protocol and a read length of 151 nt. And sequenced on a Miseq (Illumina InC, San Diego, CA, USA) platform.

The Breseq pipeline (Deerage and Barric, 2014) version 0.23 and Bowrie 2 (Langmead and Salzberg) were used to map sequencing reads and to identify sequence variants to the E. coli K12 MG1655 reference genome (NIBI accession number). NC_000913.2). According to the manual, the gene deletions present in the strains were evaluated based on the missing coverage areas in the genome. Common variants found in MG1655 stock culture (Freddolino et al., 2012) were excluded from further analysis. All sequencing samples had an average map coverage of at least 25 times. This experiment shows mutations that cause increased resistance to high concentrations of serine, as shown in Table S5.

Table S5: Mutations found in strains responsible for increased serine resistance. The reference number for the genomic sequence is NC_000913.

Explanation of symbols:
*: Insertion indicating stop codon ¹ : Insertion of insertion sequence element IS1 of 768 base pairs in length in the lagging strand at position 3966174, which is an intergenic region between trxA and rho. Overlapping 9 base pairs upstream and downstream of the insert is observed.
Insertion ² : Insertion of 1 base pair at position 2942629, an intergenic region between the genes gcvA and ygdI.
Insertion ³ : Insertion of insertion sequence element IS4 1342 base pairs long at position 2942878, an intergenic region between the genes gcvA and ygdI. Overlapping 13 base pairs upstream and downstream of the insert is observed.
Insertion ⁴ : Insertion of 1 base pair at position 2599854, an intergenic region between the genes dapA and gcvR.
Insertion ⁵ : Insertion of insertion sequence element IS1 of 768 base pairs in length in the lagging strand at position 2492323. This leads to truncation of the gene frc. Overlap is observed 9 base pairs upstream and downstream of the insert.
Deletion ⁶ : Deletion of 2854 base pairs from position 850092, a deletion of the first 5 base pairs of rhtA, a complete deletion of genes ompX and ybiP, a deletion of 239 bp of sRNA rybA and 77 of the mntS gene. This results in a base pair deletion.
Insertion ⁷ : Insertion of insertion sequence element IS5 at position 121518 with a length of 1195 base pairs, which results in the deletion of the majority of aroP.
Insertion ⁸ : Insertion of insert sequence element IS1 of 768 base pairs in length in the lagging strand at position 1673370, an intergenic region between the genes mdtJ and tqsA. Overlap is observed 9 base pairs upstream and downstream of the insert.
Intergenic ⁹ : C→T mutation at position 923321, an intergenic region between trxB and Irp.
Intergenic ¹⁰ : T→C mutation at position 4428871, an intergenic region between yftB and fkIV. The mutations are 154 base pairs upstream of yftB and 64 base pairs upstream of fklB.

Example 6-Reverse Engineering and Complex Genomic Engineering of ALE Mutations by the cam-sacB System To identify mutations that cause increased resistance to serine, two different methods, as described below, were used to demonstrate The mutation identified in 5 was inserted into strain Q1(DE3).

Introduction of A. thrA mutations Mutations in thrA were introduced into the genome of strain Q1(DE3) using a cam-sacB based selection system. The cam-sacB was inserted using the pkD46 plasmid in which the exo, β and γ genes for recombination live. Positive selection for cassette insertion was done by selecting clones for chloramphenocol resistance. Cassette loss was selected by selection of replica-plating clones on LB-sucrose plates containing LB-chloramphenocol and 15% sucrose (without NaCl). The Cam-sacB cassette was amplified using the thrA_camsacB_F and R (Table S6) primers, respectively. Aside from template and extension time, the reaction mixture and PCR program were the same as described in Example 1. The extension time was 2 minutes and 30 seconds, while the template was 1 μl of an overnight culture of an E. coli culture containing the cam-sacB cassette. Competent Q1 (DE3) cells were then transformed with 200 ng of the thrA-cam-sacB cassette and after 2 hours plated on LB-chloramphenicol-amplicon plates and incubated at 30°C overnight. .. Single colonies were picked and made electrocompetent 1 hour after induction (Example 1) and transformed with the thrA allele. Two hours after recovery, cells were plated on LB-sucrose and incubated at 37°C to recover the pkD46 plasmid. Twenty-four clones from each experiment were replica plated on LB-chloramphenicol and LB plates. Clones that did not grow on chloramphenicol plates were checked by colony PCR for cassette loss and subsequently sequenced by Sanger sequencing.

All three strains containing the thrA mutant (Y356C, S357R or S359R) were grown in M9 medium supplemented with 2 mM glycine, 2 g/L glucose and 6.25 g/L L-serine. In this experiment, background was not subtracted from the measurements, resulting in a clear increase in OD of strain Q1. However, under these conditions, strain Q1 did not show significant growth. On the other hand, each thrA mutation resulted in a similar and very marked increase in resistance to L-serine (Fig. 7), which, unlike the parental strain Q1, led to these at L-serine concentrations of at least 6.25 g/L. Enable to grow. Since all thrA mutants have the same growth profile, the S357R mutant was selected as the parent strain for complex genomic engineering described below (designated strain Q1(DE3)-thrAS357R).

Table S6: Primers used for genomic insertion of thrA mutations in the genome

B. Complex Genome Engineering, Screening, and Amplicon Sequencing of Selected ALE Mutations Complex genomic engineering (Wang et al., 2011) was used to identify mutations that cause increased resistance to serine and was demonstrated in Example 5. A selective mutation was introduced into the Q1(DE3)-thrAS357R strain.

The protocol applied to complex genome engineering was similar to the method published by Wang and Church (2011). Strain Q1(DE3)-thrAS357R was transformed with plasmid pMA1 (containing only β protein under control of arabinose inducible promoter). Clones were plated on LB-amplicons and incubated at 37°C. Colonies were cultivated overnight in TY-amp-gly medium at 37° C. and reseeded the next day in 25 ml of fresh TY-gly medium. After reaching an OD of 0.4 at 37°C, arabinose was added to a final concentration of 0.2%. Cells were re-incubated at 37° C. and 250 rpm for a further 15 minutes and then made electrocompetent by two washes with 10% glycerol (ice cold). The final volume of electrocompetent cells was adjusted to 200 μl with 10% glycerol. Primers targeting loci (IrpD143G, enoV164L, argP Q132K, cycA1220V, pykF E250 ^* , rpoB520L, gcvA ^* , yojL D334H, rho R87L) are listed in Table S7. All primers were pooled and adjusted to a final concentration of 10 pmol/μl. To 50 μl of cells 1 μl of this mixture was added and transformed by electrophoresis. The transformed cells were added directly to 25 ml TY-gly medium and incubated at 37° C., 250 rpm for 2 hours to reach an OD of 0.4, followed by a second complex engineering as described above. Arabinose induction and transformation. This process was repeated 6 times. After the sixth round of complex engineering, cells were incubated overnight. The resulting library of mutants was subsequently grown in M9 minimal medium with or without serine. Thus, the mutations that gave rise to increased growth rates or increased resistance to serine in minimal medium were increased: 1.5 μl total overnight culture supplemented with 2 mM glycine and 2 g/L glucose. Triplicates were added to wells in microtiter plates containing 150 μl triplicates of M9 medium (minimal medium) or M9 medium with 2 mM glycine, 2 g/L glucose and 25 g/L serine (minimal medium and serine). The growth profile was monitored for 40 hours at 37°C with continuous rapid shaking in a microtiter plate reader. The optical density was measured at 630 nm every 5 minutes. Triplicates were pooled and used as a template for amplicon production. The PCR program and reaction composition are described in Example 1. The Illumina protocol was followed by amplicon processing, and subsequent gene sequencing was as described in Example 5. The resulting sequencing was analyzed by computer, and the enrichment of various mutations was calculated as the frequency of mutations in either minimal medium or minimal medium with serine, and the mutations in the non-selected library (Fig. 8). This experiment shows that mutations in Irp, rho, argP and eno resulted in increased resistance to serine.

Table S7: Primers used for complex genome engineering

Table S8: Primers used for amplicon sequencing

Example 7: Serine Production by Escherichia coli Resistant and Susceptible During Fed-Batch Fermentation In order to increase expression of the exporter with the serine pathway in the pACYC-Duet vector, a new vector following the same strategy as in Example 2 above. Was built. SerB was cloned using a NcoI and HindIII double digest for cloning ydeD-C-His, followed by ligation. Next, the obtained vector (pACYC-Duet-serB) was double-digested with NcoI and PacI for cloning ydeD-C-His. Both strains Q1(DE3) and Q3(DE3) were made electrocompetent by growing the cells to mid-log phase (37° C., 250 rpm) in 30 ml of 2×YT medium supplemented with 0.2% glucose. Cells were harvested by centrifugation at 6500 rpm for 5 minutes at 4°C and washed twice with ice-cold 10% glycerol. Finally the cells were resuspended in 500 μL of 10% glycerol and 50 μL of cells were transformed with the plasmids pcDF-Duet1-serAmut-serC and pACYC, SerB-ydeD-c-His for serine production.

Serine production was demonstrated during fed-batch fermentation in a 1 L fermentor (Sartorius, Gottingen, Germany). The medium was 2 g/L MGSO ₄ *7H ₂ O, 2 g/L KH ₂ PO ₄ , 5 g/L (NH ₄ )SO ₄ , 7.5 g/L glucose, 2 g/L yeast extract, 0.6 g/L glycine. , 0.12 g/L thionine, 4x trace elements (as described in Example 2), 50 mg/L spectinomycin and 25 mg/L chloramphenicol. For the E. coli Q1 (DE3) strain, the initial glucose concentration was 10 g/L to promote growth prior to induction. The feeds are generally 140 g/L glucose, 24 g/L ammonium sulfate, 2 g/L glycine, 0.12 g/L threonine, 2.5 g/L each of MGSO ₄ *7H ₂ O and KH ₂ PO, 1×. It contained trace elements, 50 mg/L spectinomycin and 25 mg/L chloramphenicol, whereas the feed of strain Q1(DE3) contained 120 g/L glucose.

Log phase cultures were used to seed 500 ml medium at a seeding ratio of 1:50. Cultures were grown overnight at 37° C., baffle speed of 1000 rpm and 20% air saturation. A feeding rate of 8 g/h was started after the glucose concentration in the medium fell below 250 mg/L. Production was induced in the late log phase (OD 7.5-9.5) by the addition of 40 μM IPTG and the feed rate was reduced to 6 g/h. Samples were taken at regular intervals and HPLC and LCMS analyzes were performed as previously described.

This experiment demonstrates that the use of fed-batch fermentation allows serine to be produced in E. coli in high titers and yields (Figure 9). Titers were 6.8 g/L and 12.5 g/L for the Q1(DE3) and Q3(DE3) strains. Fermentation resulted in a significantly higher mass yield of 36.7% from glucose in strain Q3(DE3) compared to 24.3% in strain Q1(DE3) (FIG. 9B). This indicates that higher titers and yields can be achieved using production strains that are resistant to serine.

Example 8-Reverse Endianilized Strains Showing Increased Serine Production The reverse endianilized strains of Example 6 were checked for serine resistance as described in Example 3. The best resistant strain (F7) was checked for serine production and genomic sequenced as described in Example 5. A plasmid for overexpressing the serine production pathway was introduced as described in Example 7. Shake flask experiments were performed as described in Example 2, with the only difference being that the concentration of glucose was 2.5 g/L. O. D. Measurements and sampling were performed at regular intervals and samples were analyzed using the method described in Example 2. Surprisingly, the strain produced an increased production and yield of serine from glucose as shown in Table S9. The genomic sequence defines a deletion of zwf, which encodes the first enzyme in the pentose phosphate pathway. In addition, this strain carried the thrAS357R and rhoR87L mutations. Moreover, the strain had a truncation of the branched chain amino acid exporter brnQ (105 base pairs were deleted at the beginning of position 419986. A 439 amino acid protein was truncated after 308 amino acids).

Table S9: Effect of Δzwf on serine production

Example 9-Production by ALE8 In order to use the pET vector as an expression system, a DE3 cassette containing T7 polymerase was prepared from the ALE8-8 mutant (Example 5) using the λDE3 lysis kit (Millipore, Damstadt, Germany). Inserted into the genome, resulting in strain ALE8-8(DE3). Subsequently, ALE8-8(DE3) was made electrocompetent by growing the cells to mid-log phase (37° C., 250 rpm) in 30 ml of 2×YT medium supplemented with 0.2% glucose. Cells were harvested by centrifugation at 6500 rpm for 5 minutes at 4°C and washed twice with ice-cold 10% glycerol. Finally, the cells were resuspended in 500 μL of 10% glycerol, and 50 μL of cells were transformed with serine-producing plasmids pCDF-Duetl-serAmut-serC and pACYC, SerB.

Serine production was demonstrated during fed-batch fermentation in the 1 L fermentor described in Example 7 (Sartorius, Gottingen, Germany). The medium was 2 g/L MGSO ₄ *7H ₂ O, 2 g/L KH ₂ PO ₄ , 5 gL(NH ₄ ) ₂ SO ₄ , 10 g/L glucose, 2 g/L yeast extract, 0.6 g/L glycine, 4×. It contained trace elements (as described in Example 2), 50 mg/L spectinomycin and 25 mg/L chloramphenol. 375 g of the feed is 400 g/L glucose, 24 g/L ammonium sulfate, 2 g/L glycine, 2.5 g/L MGSO ₄ *7H ₂ O and KH ₂ PO ₄ 5 g/L, 1×trace element 50 mg/L, 50 mg /L spectinomycin and 25 mg/L chloramphenol. Log phase cultures were used to inoculate 500 ml medium with a 1:50 inoculation ratio. The culture was grown in a fermentor at 37° C., agitation speed of 1000 rpm and 20% air saturation. Production was induced in the late log phase (OD 8.5-9.5) by the addition of 80 μM IPTG and feeding was initiated at a rate of 8 g/h. Samples were taken at regular intervals and HPLC and LCMS analyzes were performed as previously described.

This experiment showed that both high cell density and high serine titer (55.0 g/L) were obtained in the ALE8-8(DE3) strain (FIG. 10A) with a mass yield from glucose of 36. It was 0.0% (FIG. 10B).

Example 10-ThrA Overexpression and Site Saturation Mutations at Positions Y356, S357 and S359 Site mutated mutations (SSM) were used to mutate selected amino acid residues and all other 20 amino acids. Can be This makes it possible to investigate the efficiency of amino acid substitutions in the enzymatic activity, and in turn the phenotype. The thrA gene was amplified from strain MG-1655wt using primers thrA_NcoI_F and thrA_HindIII (primer sequences in Table S10) and cloned in pACYC-Duetl plasmid using NcoI and HindIII enzymes. The cloning protocol was the same as the cloning of serB in Example 2. The constructed vector was pACYC-thrA. This plasmid was used as a template for SSM. The SSM for the thrA mutations observed in the ALE strains (Y356, S357 and S359) was performed using the primers listed in Table S10. The SSM protocol was similar to the SDM protocol applied in Example 2 with respect to the mutation of feedback intensive serA. The PCR product was digested with Dpnl and transformed into strain Q1(DE3), which carries the wild-type thrA gene in this genome and was supplemented with 0.1% glucose and 4 mM glycine. It was plated on a phenol plate. Chloramphenicol was supplemented in all the following media for plasmid stability.

Individual colonies were picked and plated on 96 well MTP plates containing 2xTY medium supplemented with 0.1% glucose and 2 mM glycine. 90 colonies (1 microtiter plate (MTP)) were picked from each SSM library (3 plates in total). The Q1(DE3) strain in which pACYC-Duet empty vector and pACYC-thrAwt inhabit were used as control strains and seeded in each plate. The plate was incubated overnight at 37°C and 300 rpm. This pre-culture plate was used as a seeding source from which 1 μL was added to a new 96-well MTP plate containing 100 μL of M9 medium supplemented with 0.2% glucose, 2 mM glycine. Cultures were incubated for 4 hours, after which 25 μL of 1 mM IPTG was added to induce expression. Subsequently, the plate is incubated for 2 hours and then M9 medium containing 0.2% glucose, 2 mM glycine and 12.5 g/L L-serine is added, thus giving a final serine concentration of 6.25 g/L. Reached The plates were then incubated in a MTP reader with shaking for growth analysis as previously described. Selected resistant clones from each plate were recovered in one plate and the resistance test repeated for these strains. Plasmids from resistant clones were sequenced and growth rates estimated from the exponential phase of the culture. In this way, the number of amino acid substitutions resulting in resistance to serine was identified (Fig. 11). FIG. 11(A-C) shows that the growth rates of mutant strains with various amino acid substitutions were observed at ThrA positions 356, 357 and 359, respectively (indicated by one-letter codes, respectively). ). The growth rate of E. coli carrying the wild-type thrA gene is designated as "wt".

This experiment demonstrates that amino acid substitutions at positions 356, 357 and 359 in the native ThrA protein ensured the increased resistance of the degenerate strain to L-serine. Furthermore, the data demonstrate that overexpression of mutants of ThrA can increase resistance to L-serine even in strains with the native thrA gene present in their genome.

Table S10: Primers used for thrA cloning and site saturation mutagenesis (SSM)

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Claims (13)

A bacterium belonging to the family Enterobacteriaceae which has been modified to inactivate the genes sdaA, sdaB, tdcG and glyA . Wherein the bacterium is 3- phosphoglycerate dehydrogenase, phosphoglycerate is further modified to overexpress phosphoserine phosphatase and phosphoserine aminotransferase, bacterium according to claim 1. Wherein the bacterium has also reduced L- serine 6. The bacterium according to claim 1 or 2 , which is capable of growing in a minimal medium containing a concentration of 25 g/L. Wherein the bacterium has also a small 6. The method according to any one of claims 1 to 3 , which is capable of growing at a growth rate of at least 0.1 hr ^-1 during logarithmic growth in minimal medium containing L-serine at a concentration of 25 g/L. Bacteria. Wherein the bacterium expresses a polypeptide having an amino acid sequence having Y356, S357 and / or least be 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11 comprising an amino acid substitution at the position of S359, the In this case, the substitution at the position of Y356 is selected from Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R; The substitution at position S357 is selected from the group consisting of S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F and S357H; and the substitution at position S359 is S359R, S359G, S359M, S359F. The bacterium according to any one of claims 1 to 4 , which is selected from the group consisting of S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L. Wherein the bacterium has a nucleotide sequence encoding a polypeptide having an amino acid sequence having Y356, S357 and / or least be 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11 comprising an amino acid substitution at the position of the S359 A bacterium according to any one of claims 1 to 5 , which comprises an exogenous nucleic acid molecule comprising Substitutions at the Y356 position are selected from Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R; The substitution at position is selected from the group consisting of S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; and the substitution at position S359 is S359R, S359G. The bacterium according to claim 6 , which is selected from the group consisting of S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L. Wherein the bacterium has the amino acid sequence set forth in SEQ ID NO: 9 contains an exogenous nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 90% sequence identity, Claims 1 to 7 The bacterium according to any one of 1. The bacterium has the following polypeptides (i) to (xv):
(I) a polypeptide having an amino acid sequence having at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 12, wherein D is substituted with another amino acid at position 143 in the amino acid sequence ,
(Ii) a polypeptide having an amino acid sequence having at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 13, wherein R is substituted with another amino acid at position 87 in the amino acid sequence ,
(Iii) A polypeptide having an amino acid sequence having a sequence identity of at least 90% with the amino acid sequence set forth in SEQ ID NO: 14, wherein V is substituted with another amino acid at position 164 in the amino acid sequence. ,
(Iv) A polypeptide having an amino acid sequence having a sequence identity of at least 90% with the amino acid sequence of SEQ ID NO: 15, wherein Q is substituted with another amino acid at position 132 in the amino acid sequence. ,
(V) a polypeptide having an amino acid sequence having a sequence identity of at least 90% with the amino acid sequence set forth in SEQ ID NO: 16, wherein G is replaced with another amino acid at position 19 in the amino acid sequence ,
(Vi) A polypeptide having an amino acid sequence having at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 17, wherein I is substituted with another amino acid at position 220 in the amino acid sequence. ,
(Vii) a polypeptide having an amino acid sequence having at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 18, wherein I is substituted with another amino acid at position 202 in the amino acid sequence ,
(Viii) A polypeptide having an amino acid sequence having at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 19, wherein D is substituted with another amino acid at position 334 in the amino acid sequence. ,
(Ix) A polypeptide having an amino acid sequence having at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 20, wherein V is substituted with another amino acid at position 120 in the amino acid sequence. ,
(X) a polypeptide having an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:22,
(Xi) a polypeptide having an amino acid sequence having at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 24,
(Xii) a polypeptide having an amino acid sequence having at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 25, wherein P is substituted with another amino acid at position 520 in the amino acid sequence ,
(Xiii) a polypeptide having an amino acid sequence having at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 26, wherein T is substituted with another amino acid at position 218 in the amino acid sequence ,
(Xiv) A polypeptide having an amino acid sequence having at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 27, wherein A is substituted with another amino acid at position 178 in the amino acid sequence. ,
And/or
(Xv) A polypeptide having an amino acid sequence having at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 29.
The bacterium according to any one of claims 1 to 8 , which expresses any one of the above. The bacterium according to any one of claims 1 to 9 , wherein the bacterium belongs to the genus Escherichia coli. The bacterium according to any one of claims 1 to 10 , wherein the bacterium is Escherichia coli. A method for producing L-serine or an L-serine derivative, the method comprising culturing the bacterium according to any one of claims 1 to 11 in a medium. Manufacturing method. 13. The method of claim 12 , wherein the L-serine derivative is selected from the group consisting of L-cysteine, L-methionine, L-glycine, O-acetylserine, L-tryptophan, thiamine, ethanolamine and ethylene glycol.

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